Cysteine variants of granulocyte colony-stimulating factor

ABSTRACT

The growth hormone supergene family comprises greater than 20 structurally related cytokines and growth factors. A general method is provided for creating site-specific, biologically active conjugates of these proteins. The method involves adding cysteine residues to non-essential regions of the proteins or substituting cysteine residues for non-essential amino acids in the proteins using site-directed mutagenesis and then covalently coupling a cysteine-reactive polymer or other type of cysteine-reactive moiety to the proteins via the added cysteine residue. Disclosed herein are preferred sites for adding cysteine residues or introducing cysteine substitutions into the proteins, and the proteins and protein derivatives produced thereby.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.10/400,377, filed Mar. 26, 2003, which is a divisional of U.S.application Ser. No. 09/462,941, filed Jan. 14, 2000, now U.S. Pat. No.6,608,183, which is a national stage application under 35 U.S.C. § 371of PCT Application Serial No. PCT/US98/14497, filed Jul. 13, 1998, whichclaims the benefit of priority from U.S. Provisional Application Ser.No. 60/052,516, filed Jul. 14, 1997, each of which is incorporatedherein by reference in its entirety.

GOVERNMENT SUPPORT

[0002] This invention was made in part with government support underGrant Nos. 1R43 CA78094-01 and 2R44 CA78094-02, each awarded by theNational Institutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

[0003] The present invention relates to genetically engineeredtherapeutic proteins. More specifically, the engineered proteins includegrowth hormone and related proteins.

BACKGROUND OF THE INVENTION

[0004] The following proteins are encoded by genes of the growth hormone(GH) supergene family (Bazan (1990); Mott and Campbell (1995);Silvennoinen and lhle (1996)): growth hormone, prolactin, placentallactogen, erythropoietin (EPO), thrombopoietin (TPO), interleukin-2(IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35subunit) IL-13, IL-15, oncostatin M, ciliary neurotrophic factor,leukemia inhibitory factor, alpha interferon, beta interferon, gammainterferon, omega interferon, tau interferon, granulocyte-colonystimulating factor (G-CSF), granulocyte-macrophage colony stimulatingfactor (GM-CSF), macrophage colony stimulating factor (M-CSF) andcardiotrophin-1 (CT-1) (“the GH supergene family”). It is anticipatedthat additional members of this gene family will be identified in thefuture through gene cloning and sequencing. Members of the GH supergenefamily have similar secondary and tertiary structures, despite the factthat they generally have limited amino acid or DNA sequence identity.The shared structural features allow new members of the gene family tobe readily identified.

[0005] There is considerable interest on the part of patients andhealthcare providers in the development of long acting, “user-friendly”protein therapeutics. Proteins are expensive to manufacture and, unlikeconventional small molecule drugs, are not readily absorbed by the body.Moreover, they are digested if taken orally. Therefore, natural proteinsmust be administered by injection. After injection, most proteins arecleared rapidly from the body, necessitating frequent, often daily,injections. Patients dislike injections, which leads to reducedcompliance and reduced drug efficacy. Some proteins, such aserythropoietin (EPO), are effective when administered less often (threetimes per week for EPO) because they are glycosylated. However,glycosylated proteins are produced using expensive mammalian cellexpression systems.

[0006] The length of time an injected protein remains in the body isfinite and is determined by, e.g., the protein's size and whether or notthe protein contains covalent modifications such as glycosylation.Circulating concentrations of injected proteins change constantly, oftenby several orders of magnitude, over a 24-hour period. Rapidly changingconcentrations of protein agonists can have dramatic downstreamconsequences, at times under-stimulating and at other timesover-stimulating target cells. Similar problems plague proteinantagonists. These fluctuations can lead to decreased efficacy andincreased frequency of adverse side effects for protein therapeutics.The rapid clearance of recombinant proteins from the body significantlyincreases the amount of protein required per patient and dramaticallyincreases the cost of treatment. The cost of human proteinpharmaceuticals is expected to increase dramatically in the years aheadas new and existing drugs are approved for more disease indications.

[0007] Thus, there is a need to develop protein delivery technologiesthat lower the costs of protein therapeutics to patients and healthcareproviders. The present invention provides a solution to this problem byproviding methods to prolong the circulating half-lives of proteintherapeutics in the body so that the proteins do not have to be injectedfrequently. This solution also satisfies the needs and desires ofpatients for protein therapeutics that are “user-friendly”, i.e.,protein therapeutics that do not require frequent injections. Thepresent invention solves these and other problems by providingbiologically active, cysteine-added variants of members of the growthhormone supergene family. The invention also provides for the chemicalmodification of these variants with cysteine-reactive polymers or othertypes of cysteine-reactive moieties to produce derivatives thereof andthe molecules so produced.

SUMMARY OF THE INVENTION

[0008] The present invention provides cysteine variants of members ofthe GH supergene family. The variants comprise a cysteine residuesubstituted for a nonessential amino acid of the proteins. Preferably,the variants comprise a cysteine residue substituted for an amino acidselected from amino acids in the loop regions, the ends of the alphahelices, proximal to the first amphipathic helix, and distal to thefinal amphipathic helix or wherein the cysteine residue is added at theN-terminus or C-terminus of the proteins. Preferred sites forsubstitution are the N— and O-linked glycosylation sites.

[0009] Also provided are cysteine variants wherein the amino acidsubstituted for is in the A-B loop, B-C loop, the C-D loop or D-E loopof interferon/interferon-10-like members of the GH supergene family.

[0010] Also provided are cysteine variants of members of the GHsupergene family wherein the cysteine residue is introduced between twoamino acids in the natural protein. In particular, the cysteine residueis introduced into the loop regions, the ends of the alpha helices,proximal to the first amphipathic helix, or distal to the finalamphipathic helix. Even more particularly, the cysteine variant isintroduced between two amino acids in an N—O-linked glycosylation siteor adjacent to an amino acid in an N-linked or O-linked glycosylationsite.

[0011] More particularly are provided cysteine variants wherein the loopregion where the cysteine is introduced is the A-B loop, the B-C loop,the C-D loop or D-E loop of interferon/interferon-10-like members of theGH supergene family.

[0012] Such cysteine substitutions or insertion mutations also caninclude the insertion of one or more additional amino acids amino acidsat the amino-terminal or carboxy-terminal to the cysteine substitutionor insertion.

[0013] Also provided are cysteine variants that are further derivatisedby PEGylating the cysteine variants and including the derivatisedproteins produced thereby.

[0014] As set forth in the examples, specific cysteine variants of themembers of the GH supergene family also are provided, including forexample, variants of GH. The GH cysteine variants can have thesubstituted-for amino acid or inserted cysteine located at theN-terminal end of the A-B loop, the B-C loop, the C-D loop, the firstthree or last three amino acids in the A, B, C and D helices and theamino acids proximal to helix A and distal to helix D.

[0015] More particularly, the cysteine can be substituted for thefollowing amino acids: F1, T3, P5, E33, A34, K38, E39, Q40, S43, Q46,N47, P48, Q49, T50, S51, S55, T60, A98, N99, S100, G104, A105, S106,E129, D130, G131, S132, P133, T135, G136, Q137, K140, Q141, T142, S144,K145, D147, T148, N149, S150, H151, N152, D153, S184, E186, G187, S188,and G190.

[0016] Other examples of cysteine variants according to the inventioninclude erythropoietin variants. Erythropoietin variants include thosewherein the substituted for amino acid is located in the A-B loop, theB-C loop, the C-D loop, the amino acids proximal to helix A and distalto helix D and the N— or C-terminus. Even more specifically, the EPOcysteine variants include molecules wherein the amino acids indicatedbelow have a cysteine substituted therefor: serine-126, N24, I25, T26,N38, I39, T40, N83, S84, A1, P2, P3, R4, D8, S9, T27, G28, A30, E31,H32, S34, N36, D43, T44, K45, N47, A50, K52, E55, G57, Q58, G77, Q78,A79, Q86, W88, E89, T107, R110, A111, G113, A114, Q15, K116, E117, A118,S120, P121, P122, D123, A124, A125, A127, A128, T132, K154, T157, G158,E159, A160, T163, G164, D165, R166 and S85.

[0017] The members of the GH supergene family include growth hormone,prolactin, placental lactogen, erythropoietin, thrombopoietin,interleukin-2, interleukin-3, interleukin-4, interleukin-5,interleukin-6, interleukin-7, interleukin-9, interleukin-10,interleukin-1, interleukin-12 (p35 subunit), interleukin-13,interleukin-15, oncostatin M, ciliary neurotrophic factor, leukemiainhibitory factor, alpha interferon, beta interferon, gamma interferon,omega interferon, tau interferon, granulocyte-colony stimulating factor,granulocyte-macrophage colony stimulating factor, macrophage colonystimulating factor, cardiotrophin-1 and other proteins identified andclassified as members of the family. The proteins can be derived fromany animal species including human, companion animals and farm animals.

[0018] Other variations and modifications to the invention will beobvious to those skilled in the art based on the specification and the“rules” set forth herein. All of these are considered as part of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention relates to cysteine variants and, amongother things, the site-specific conjugation of such proteins withpolyethylene glycol (PEG) or other such moieties. PEG is anon-antigenic, inert polymer that significantly prolongs the length oftime a protein circulates in the body. This allows the protein to beeffective for a longer period of time. Covalent modification of proteinswith PEG has proven to be a useful method to extend the circulatinghalf-lives of proteins in the body (Abuchowski et al., 1984; Hershfield,1987; Meyers et al., 1991). Covalent attachment of PEG to a proteinincreases the protein's effective size and reduces its rate of clearancerate from the body. PEGs are commercially available in several sizes,allowing the circulating half-lives of PEG-modified proteins to betailored for individual indications through use of different size PEGs.Other benefits of PEG modification include an increase in proteinsolubility, an increase in in vivo protein stability and a decrease inprotein immunogenicity (Katre et al., 1987; Katre, 1990).

[0020] The preferred method for PEGylating proteins is to covalentlyattach PEG to cysteine residues using cysteine-reactive PEGs. A numberof highly specific, cysteine-reactive PEGs with different reactivegroups (e.g., maleimide, vinylsulfone) and different size PEGs (2-20kDa) are commercially available (e.g., from Shearwater, Polymers, Inc.,Huntsville, Ala.). At neutral pH, these PEG reagents selectively attachto “free” cysteine residues, i.e., cysieine residues not involved indisulfide bonds. The conjugates are hydrolytically stable. Use ofcysteine-reactive PEGs allows the development of homogeneous PEG-proteinconjugates of defined structure.

[0021] Considerable progress has been made in recent years indetermining the structures of commercially important proteintherapeutics and understanding how they interact with their proteintargets, e.g., cell-surface receptors, proteases, etc. This structuralinformation can be used to design PEG-protein conjugates usingcysteine-reactive PEGs. Cysteine residues in most proteins participatein disulfide bonds and are not available for PEGylation usingcysteine-reactive PEGs. Through in vitro mutagenesis using recombinantDNA techniques, additional cysteine residues can be introduced anywhereinto the protein. The added cysteines can be introduced at the beginningof the protein, at the end of the protein, between two amino acids inthe protein sequence or, preferably, substituted for an existing aminoacid in the protein sequence. The newly added “free” cysteines can serveas sites for the specific attachment of a PEG molecule usingcysteine-reactive PEGs. The added cysteine must be exposed on theprotein's surface and accessible for PEGylation for this method to besuccessful. If the site used to introduce an added cysteine site isnon-essential for biological activity, then the PEGylated protein willdisplay essentially wild type (normal) in vitro bioactivity. The majortechnical challenge in PEGylating proteins with cysteine-reactive PEGsis the identification of surface exposed, non-essential regions in thetarget protein where cysteine residues can be added or substituted forexisting amino acids without loss of bioactivity.

[0022] Cysteine-added variants of a few human proteins and PEG-polymerconjugates of these proteins have been described. U.S. Pat. No.5,206,344 describes cysteine-added variants of IL-2. Thesecysteine-added variants are located within the first 20 amino acids fromthe amino terminus of the mature IL-2 polypeptide chain. The preferredcysteine variant is at position 3 of the mature polypeptide chain, whichcorresponds to a threonine residue that is O-glycosylated in thenaturally occurring protein. Substitution of cysteine for threonine atposition 3 yields an IL-2 variant that can be PEGylated with acysteine-reactive PEG and retain full in vitro bioactivity (Goodson andKatre, 1990). In contrast, natural IL-2 PEGylated with lysine-reactivePEGs displays reduced in vitro bioactivity (Goodson and Katre, 1990).The effects of cysteine substitutions at other positions in IL-2 werenot reported.

[0023] U.S. Pat. No. 5,166,322 teaches cysteine added variants of IL-3.This variants are located within the first 14 amino acids from theN-terminus of the mature protein sequence. The patent teaches expressionof the proteins in bacteria and covalent modification of the proteinswith cysteine-reactive PEGs. No information is provided as to whetherthe cysteine-added variants and PEG-conjugates of IL-3 are biologicallyactive. Cysteine-added variants at other positions in the polypeptidechain were not reported.

[0024] World patent application WO9412219 and PCT application US95/06540teach cysteine-added variants of insulin-like growth factor-I (IGF-I).IGF-I has a very different structures from GH and is not a member of theGH supergene family (Mott and Campbell, 1995). Cysteine substitutions atmany positions in the IGF-I protein are described. Only certain of thecysteine-added variants are biologically active. The preferred site forthe cysteine added variant is at amino acid position 69 in the matureprotein chain. Cysteine substitutions at positions near the N-terminusof the protein (residues 1-3) yielded IGF-I variants with reducedbiological activities and improper disulfide bonds.

[0025] World patent application WO9422466 teaches two cysteine-addedvariants of insulin-like growth factor (IGF) binding protein-1, whichhas a very different structure than GH and is not a member of the GHsupergene family. The two cysteine-added IGF binding protein-1 variantsdisclosed are located at positions 98 and 101 in the mature proteinchain and correspond to serine residues that are phosphorylated in thenaturally-occurring protein.

[0026] U.S. patent application Ser. No. 07/822296 teaches cysteine addedvariants of tumor necrosis factor binding protein, which is a soluble,truncated form of the tumor necrosis factor cellular receptor. Tumornecrosis factor binding protein has a very different structure than GHand is not a member of the GH supergene family.

[0027] IGF-I, IGF binding protein-1 and tumor necrosis factor bindingprotein have secondary and tertiary structures that are very differentfrom GH and the proteins are not members of the GH supergene family.Because of this, it is difficult to use the information gained fromstudies of IGF-I, IGF binding protein-l and tumor necrosis factorbinding protein to create cysteine-added variants of members of the GHsupergene family. The studies with IL-2 and IL-3 were carried out beforethe structures of IL-2 and IL-3 were known (McKay 1992; Bazan, 1992) andbefore it was known that these proteins are members of the GH supergenefamily. Previous experiments aimed at identifying preferred sites foradding cysteine residues to IL-2 and IL-3 were largely empirical andwere performed prior to experiments indicating that members of the GHsupergene family possessed similar secondary and tertiary structures.

[0028] Based on the structural information now available for members ofthe GH supergene family, the present invention provides “rules” fordetermining a priori which regions and amino acid residues in members ofthe GH supergene family can be used to introduce or substitute cysteineresidues without significant loss of biological activity. In contrast tothe naturally occurring proteins, these cysteine-added variants ofmembers of the GH supergene family will possess novel properties such asthe ability to be covalently modified at defined sites within thepolypeptide chain with cysteine-reactive polymers or other types ofcysteine-reactive moieties. The covalently modified proteins will bebiologically active.

[0029] GH is the best-studied member of the GH supergene family. GH is a22 kDa protein secreted by the pituitary gland. GH stimulates metabolismof bone, cartilage and muscle and is the body's primary hormone forstimulating somatic growth during childhood. Recombinant human GH (rhGH)is used to treat short stature resulting from GH inadequacy and renalfailure in children. GH is not glycosylated and can be produced in afully active form in bacteria. The protein has a short in vivo half-lifeand must be administered by daily subcutaneous injection for maximumeffectiveness (MacGillivray et al., 1996). Recombinant human GH (rhGH)was approved recently for treating cachexia in AIDS patients and isunder study for treating cachexia associated with other diseases.

[0030] The sequence of human GH is well known (see, e.g., Martial et al.1979; Goeddel et al. 1979 which are incorporated herein by reference;SEQ ID NO:1). GH is closely related in sequence to prolactin andplacental lactogen and these three proteins were considered originallyto comprise a small gene family. The primary sequence of GH is highlyconserved among animal species (Abdel-Meguid et al., 1987), consistentwith the protein's broad species cross-reactivity. The three dimensionalfolding pattern of porcine GH has been solved by X-ray crystallography(Abdel-Meguid et al., 1987). The protein has a compact globularstructure, comprising four amphipathic alpha helical bundles joined byloops. Human GH has a similar structure (de Vos et al., 1992). The fouralpha helical regions are termed A-D beginning from the N-terminus ofthe protein. The loop regions are referred to by the helical regionsthey join, e.g., the A-B loop joins helical bundles A and B. The A-B andC-D loops are long, whereas the B-C loop is short. GH contains fourcysteine residues, all of which participate in disulfide bonds. Thedisuifide assignments are cysteine53 joined to cysteine165 andcysteine182 joined to cysteine189.

[0031] The crystal structure of GH bound to its receptor revealed thatGH has two receptor binding sites and binds two receptor molecules(Cunningham et al., 1991; de Vos et al., 1992). The two receptor bindingsites are referred to as site I and site II. Site I encompasses theCarboxy (C)-terminal end of helix D and parts of helix A and the A-Bloop, whereas site II encompasses the Amino (N)-terminal region of helixA and a portion of helix C. Binding of GH to its receptor occurssequentially, with site I always binding first. Site II then engages asecond GH receptor, resulting in receptor dimerization and activation ofthe intracellular signaling pathways that lead to cellular responses toGH. A GH mutein in which site II has been mutated (a glycine to argininemutation at amino acid 120) is able to bind a single GH receptor, but isunable to dimerize GH receptors; this mutein acts as a GH antagonist invitro, presumably by occupying GH receptor sites without activatingintracellular signaling pathways (Fuh et al., 1992).

[0032] The roles of particular regions and amino acids in GH receptorbinding and intracellular signaling also have been studied usingtechniques such as mutagenesis, monoclonal antibodies and proteolyticdigestion. The first mutagenesis experiments entailed replacing entiredomains of GH with similar regions of the closely related protein,prolactin (Cunningham et al., 1989). One finding was that replacement ofthe B-C loop of GH with that of prolactin did not affect binding of thehybrid GH protein to a soluble form of the human GH receptor, implyingthat the B-C loop was non-essential for receptor binding. Alaninescanning mutagenesis (replacement of individual amino acids withalanine) identified 14 amino acids that are critical for GH bioactivity(Cunningham and Wells, 1989). These amino acids are located in thehelices A, B, C, and D and the A-B loop and correspond to sites I and IIidentified from the structural studies. Two lysine residues at aminoacid positions 41 and 172, K41 and K172, were determined to be criticalcomponents of the site I receptor binding site, which explains thedecrease in bioactivity observed when K1 72 is acetylated (Teh andChapman, 1988). Modification of K168 also significantly reduced GHreceptor binding and bioactivity (de la Llosa et al., 1985; Martal etal., 1985; Teh and Chapman, 1988). Regions of GH responsible for bindingthe GH receptor have also been studied using monoclonal antibodies(Cunningham et al., 1989). A series of eight monoclonal antibodies wasgenerated to human GH and analyzed for the ability to neutralize allowedthe putative binding site for each monoclonal antibody to be localizedwithin the GH three-dimensional structure. Of interest was thatmonoclonal antibodies 1 and 8 were unable to displace GH from bindingits receptor. The binding sites for these monoclonal antibodies werelocalized to the B-C loop (monoclonal number 1) and the N-terminal endof the A-B loop (monoclonal number 8). No monoclonals were studied thatbound the C-D loop specifically. The monoclonal antibody studies suggestthat the B-C loop and N-terminal end of the A-B loop are non-essentialfor receptor binding. Finally, limited cleavage of GH with trypsin wasfound to produce a two chain derivative that retained full activity(Mills et al., 1980; Li, 1982). Mapping studies indicated that trypsincleaved and/or deleted amino acids between positions 134 and 149, whichcorresponds to the C-D loop. These studies suggest the C-D loop is notinvolved in receptor binding or GH bioactivity.

[0033] Structures of a number of cytokines, including G-CSF (Hill etal., 1993), GM-CSF (Diederichs et al., 1991; Walter et al., 1992), IL-2(Bazan, 1992; McKay, 1992), IL-4 ( Redfield et al., 1991; Powers et al.,1992), and IL-5 (Milburn et al., 1993) have been determined by X-raydiffraction and NMR studies and show striking conservation with the GHstructure, despite a lack of significant primary sequence homology. EPOis considered to be a member of this family based upon modeling andmutagenesis studies (Boissel et al., 1993; Wen et al., 1994). A largenumber of additional cytokines and growth factors including ciliaryneurotrophic factor (CNTF), leukemia inhibitory factor (LIEF),thrombopoietin (TPO), oncostatin M, macrophage colony stimulating factor(M-CSF), IL-3, IL-6, IL-7, IL-9, IL-12, IL-13, IL-15, and alpha, beta,omega, tau and gamma interferon belong to this family (reviewed in Mottand Campbell, 1995; Silvennoinen and Ihle 1996). All of the abovecytokines and growth factors are now considered to comprise one largegene family, of which GH is the prototype.

[0034] In addition to sharing similar secondary and tertiary structures,members of this family share the property that they must oligomerizecell surface receptors to activate intracellular signaling pathways.Some GH family members, e.g., GH and EPO, bind a single type of receptorand cause it to form homodimers. Other family members, e.g., IL-2, IL-4,and IL-6, bind more than one type of receptor and cause the receptors toform heterodimers or higher order aggregates (Davis et al., 1993;Paonessa et al., 1995; Mott and Campbell, 1995). Mutagenesis studieshave shown that, like GH, these other cytokines and growth factorscontain multiple receptor binding sites, typically two, and bind theircognate receptors sequentially (Mott and Campbell, 1995; Matthews etal., 1996). Like GH, the primary receptor binding sites for these otherfamily members occur primarily in the four alpha helices and the A-Bloop (reviewed in Mott and Campbell, 1995). The specific amino acids inthe helical bundles that participate in receptor binding differ amongstthe family members (Mott and Campbell, 1995). Most of the cell surfacereceptors that interact with members of the GH supergene family arestructurally related and comprise a second large multi-gene family(Bazan, 1990; Mott and Campbell, 1995; Silvennoinen and Ihle 1996).

[0035] A general conclusion reached from mutational studies of variousmembers of the GH supergene family is that the loops joining the alphahelices generally tend to not be involved in receptor binding. Inparticular the short B-C loop appears to be non-essential for receptorbinding in most, if not all, family members. For this reason, the B-Cloop is a preferred region for introducing cysteine substitutions inmembers of the GH supergene family. The A-B loop, the B-C loop, the C-Dloop (and D-E loop of interferon/IL-10-like members of the GHsuperfamily) also are preferred sites for introducing cysteinemutations. Amino acids proximal to helix A and distal to the final helixalso tend not to be involved in receptor binding and also are preferredsites for introducing cysteine substitutions. Certain members of the GHfamily, e.g., EPO, IL-2, IL-3, IL-4, IL-6, G-CSF, GM-CSF, TPO, IL-10,IL-12 p35, IL-13, IL-15 and beta-interferon contain N-linked andO-linked sugars. The glycosylation sites in the proteins occur almostexclusively in the loop regions and not in the alpha helical bundles.Because the loop regions generally are not involved in receptor bindingand because they are sites for the covalent attachment of sugar groups,they are preferred sites for introducing cysteine substitutions into theproteins. Amino acids that comprise the N— and O-linked glycosylationsites in the proteins are preferred sites for cysteine substitutionsbecause these amino acids are surface-exposed, the natural protein cantolerate bulky sugar groups attached to the proteins at these sites andthe glycosylation sites tend to be located away from the receptorbinding sites.

[0036] Many additional members of the GH gene family are likely to bediscovered in the future. New members of the GH supergene family can beidentified through computer-aided secondary and tertiary structureanalyses of the predicted protein sequences. Members of the GH supergenefamily will possess four or five amphipathic helices joined bynon-helical amino acids (the loop regions). The proteins may contain ahydrophobic signal sequence at their N-terminus to promote secretionfrom the cell. Such later discovered members of the GH supergen familyalso are included within this invention.

[0037] The present invention provides “rules” for creating biologicallyactive cysteine-added variants of members of the GH supergene family.These “rules” can be applied to any existing or future member of the GHsupergene family. The cysteine-added variants will posses novelproperties not shared by the naturally occurring proteins. Mostimportantly, the cysteine added variants will possess the property thatthey can be covalently modified with cysteine-reactive polymers or othertypes of cysteine-reactive moieties to generate biologically activeproteins with improved properties such as increased in vivo half-life,increased solubility and improved in vivo efficacy.

[0038] Specifically, the present invention provides biologically activecysteine variants of members of the GH supergene family by substitutingcysteine residues for non-essential amino acids in the proteins.Preferably, the cysteine residues are substituted for amino acids thatcomprise the loop regions, for amino acids near the ends of the alphahelices and for amino acids proximal to the first amphipathic helix ordistal to the final amphipathic helix of these proteins. Other preferredsites for adding cysteine residues are at the N-terminus or C-terminusof the proteins. Cysteine residues also can be introduced between twoamino acids in the disclosed regions of the polypeptide chain. Thepresent invention teaches that N— and O-linked glycosylation sites inthe proteins are preferred sites for introducing cysteine substitutionseither by substitution for amino acids that make up the sites or, in thecase of N-linked sites, introduction of cysteines therein. Theglycosylation sites can be serine or threonine residues that areO-glycosylated or asparagine residues that are N-glycosylated. N-linkedglycosylation sites have the general structure asparagine-X-serine orthreonine (N—X—S/T), where X can be any amino acid. The asparagineresidue, the amino acid in the X position and the serine/threonineresidue of the N-linked glycosylation site are preferred sites forcreating biologically active cysteine-added variants of these proteins.Amino acids immediately surrounding or adjacent to the O-linked andN-linked glycosylation sites (within about 10 residues on either side ofthe glycosylation site) are preferred sites for introducingcysteine-substitutions.

[0039] More generally, certain of the “rules” for identifying preferredsites for creating biologically active cysteine-added protein variantscan be applied to any protein, not just proteins that are members of theGH supergene family. Specifically, preferred sites for creatingbiologically active cysteine variants of proteins (other than IL-2) areO-linked glycosylation sites. Amino acids immediately surrounding theO-linked glycosylation site (within about 10 residues on either side ofthe glycosylation site) also are preferred sites. N-linked glycosylationsites, and the amino acid residues immediately adjacent on either sideof the glycosylation site (within about 10 residues of the N—X—S/T site)also are preferred sites for creating cysteine added protein variants.Amino acids that can be replaced with cysteine without significant lossof biological activity also are preferred sites for creatingcysteine-added protein variants. Such non-essential amino acids can beidentified by performing cysteine-scanning mutagenesis on the targetprotein and measuring effects on biological activity. Cysteine-scanningmutagenesis entails adding or substituting cysteine residues forindividual amino acids in the polypeptide chain and determining theeffect of the cysteine substitution on biological activity. Cysteinescanning mutagenesis is similar to alanine-scanning mutagenesis(Cunningham et al., 1992), except that target amino acids areindividually replaced with cysteine rather than alanine residues.

[0040] Application of the “rules” to create cysteine-added variants andconjugates of protein antagonists also is contemplated. Excessproduction of cytokines and growth factors has been implicated in thepathology of many inflammatory conditions such as rheumatoid arthritis,asthma, allergies and wound scarring. Excess production of GH has beenimplicated as a cause of acromegaly. Certain growth factors andcytokines, e.g., GH and IL-6, have been implicated in proliferation ofparticular cancers. Many of the growth factors and cytokines implicatedin inflammation and cancer are members of the GH supergene family. Thereis considerable interest in developing protein antagonists of thesemolecules to treat these diseases. One strategy involves engineering thecytokines and growth factors so that they can bind to, but notoligomerize receptors. This is accomplished by mutagenizing the secondreceptor binding site (site II) on the molecules. The resulting muteinsare able to bind and occupy receptor sites but are incapable ofactivating intracellular signaling pathways. This strategy has beensuccessfully applied to GH to make a GH antagonist (Cunningham et al.,1992). Similar strategies are being pursued to develop antagonists ofother members of the GH supergene family such as IL-2 (Zurawski et al.,1990; Zurawski and Zurawski, 1992), IL-4 (Kruse et al., 1992), IL-5(Tavernier et al., 1995), GM-CSF (Hercus et al., 1994) and EPO (Matthewset al., 1996). Since the preferred sites for adding cysteine residues tomembers of the GH supergene family described here lie outside of thereceptor binding sites in these proteins, and thus removed from anysites used to create protein antagonists, the cysteine-added variantsdescribed herein could be used to generate long-acting versions ofprotein antagonists. As an example, Cunningham et al. (1992) developedan in vitro GH antagonist by mutating a glycine residue (amino acid 120)to an arginine. This glycine residue is a critical component of thesecond receptor binding site in GH; when it is replaced with arginine,GH cannot dimerize receptors. The glycine to arginine mutation atposition 120 can be introduced into DNA sequences encoding thecysteine-added variants of GH contemplated herein to create acysteine-added GH antagonist that can be conjugated withcysteine-reactive PEGs or other types of cysteine-reactive moieties.Similarly, amino acid changes in other proteins that turn the proteinsfrom agonists to antagonists could be incorporated into DNA sequencesencoding cysteine-added protein variants described herein. Considerableeffort is being spent to identify amino acid changes that convertprotein agonists to antagonists. Hercus et al.(1994) reported thatsubstituting arginine or lysine for glutamic acid at position 21 in themature GM-CSF protein converts GM-CSF from an agonist to an antagonist.Tavernier et al.(1995) reported that substituting glutamine for glutamicacid at position 13 of mature IL-5 creates an IL-5 antagonist.

[0041] Experimental strategies similar to those described above can beused to create cysteine-added variants (both agonists and antagonists)of members of the GH supergene family derived from various animals. Thisis possible because the primary amino acid sequences and structures ofcytokines and growth factors are largely conserved between human andanimal species. For this reason, the “rules” disclosed herein forcreating biologically active cysteine-added variants of members of theGH supergene family will be useful for creating biologically activecysteine-added variants of members of the GH supergene family ofcompanion animals (e.g., dogs, cats, horses) and commercial animal(e.g., cow, sheep, pig) species. Conjugation of these cysteine-addedvariants with cysteine-reactive PEGs will create long-acting versions ofthese proteins that will benefit the companion animal and commercialfarm animal markets.

[0042] Proteins that are members of the GH supergene family(hematopoietic cytokines) are provided in Silvennoimem and Ihle (1996).Silvennoimem and lhle (1996) also provide information about thestructure and expression of these proteins. DNA sequences, encoded aminoacids and in vitro and in vivo bioassays for the proteins describedherein are described in Aggarwal and Gutterman (1992; 1996), Aggarwal(1998), and Silvennoimem and lhle (1996). Bioassays for the proteinsalso are provided in catalogues of various commercial suppliers of theseproteins such as R&D Systems, Inc. and Endogen, Inc.

[0043] The following examples are provided to demonstrate how these“rules” can be used to create cysteine-added variants of GH,erythropoietin, alpha interferon, beta interferon, G-CSF, GM-CSF andother members of the GH supergene family. The examples are not intendedto be limiting, but only exemplary of specific embodiments of theinvention.

EXAMPLE 1 Cysteine-Added Variants of GH

[0044] This example discloses certain amino acids in GH that arenon-essential for biological activity and which, when mutated tocysteine residues, will not alter the normal disulfide binding patternand overall conformation of the molecule. These amino acids are locatedat the N-terminal end of the A-B loop (amino acids 34-52 of the matureprotein sequence; SEQ ID NO: 1; Martial et al 1979; Goeddel et al 1979),the B-C loop (amino acids 97-105 in the mature protein sequence), andthe C-D loop (amino acids 130-153 in the mature protein sequence). Alsoidentified as preferred sites for introducing cysteine residues are thefirst three or last three amino acids in the A, B, C and D helices andthe amino acids proximal to helix A and distal to helix D.

[0045] DNA sequences encoding wild type GH can be amplified using thepolymerase chain reaction technique from commercially availablesingle-stranded cDNA prepared from human pituitaries (ClonTech, SanDiego, Calif.) or assembled using overlapping oligonucleotides. Specificmutations can be introduced into the GH sequence using a variety ofprocedures such as phage techniques (Kunkel et al 1987), PCR mutagenesistechniques (Innis et al 1990; White 1993) mutagenesis kits such as thosesold by Stratagene (“Quick-Change Mutagenesis” kit, San Diego, Calif.)or Promega (Gene Editor Kit, Madison Wis.).

[0046] Cysteine substitutions can be introduced into any of the aminoacids comprising the B-C loop, C-D loop and N-terminal end of the A-Bloop or into the first three amino acids of the alpha helical regionsthat adjoin these regions or in the region proximal to helix A or distalto helix D. Preferred sites for introduction of cysteine residues are:F1, T3, P5, E33, A34, K38, E39, Q40, S43, Q46, N47, P48, Q49, T50, S51,S55, T60, A98, N99, S100, G104, A105, S106, E129, D130, G131, S132,P133, T135, G136, Q137, K140, Q141, T142, S144, K145, D147, T148, N149,S150, H151, N152, D153, S184, E186, G187, S188, and G190. Cysteineresidues also can be introduced at the beginning of the mature protein,i.e., proximal to the F1 amino acid, or following the last amino acid inthe mature protein, i.e., following F191. If desirable, two or more suchmutations can be readily combined in the same protein either by in vitroDNA recombination of cloned mutant genes and/or sequential constructionof individual desired mutations.

[0047] 1. Cloning the Gene for human Growth Hormone (GH)

[0048] The human GH gene was amplified from human pituitarysingle-stranded cDNA (commercially available from CLONTECH, Inc., PaloAlto, Calif.) using the polymerase chain reaction (PCR) technique andprimers BB1 and BB2. The sequence of BB1 is5′-GGGGGTCGACCATATGTTCCCAACCATTCCCTTATCCAG-3′ (SEQ ID NO: 24). Thesequence of BB2 is 5′-GGGGGATCCTCACTAGAAGCCACAGCTGCCCTC-3′ (SEQ ID NO:25). Primer BB1 was designed to encode an initiator methionine precedingthe first amino acid of mature GH, phenylalanine, and SalI and NdeIsites for cloning purposes. The reverse primer, BB2, contains a BamHIsite for cloning purposes. The PCR 100 microliter reactions contained 20pmoles of each oligonucleotide primer, 1× PCR buffer (Perkin-Elmerbuffer containing MgCl₂), 200 micromolar concentration of each of thefour nucleotides dA, dC, dG and dT, 2 ng of single-stranded cDNA, 2.5units of Taq polymerase (Perkin-Elmer) and 2.5 units of Pfu polymerase(Stratagene, Inc). The PCR reaction conditions were 96° C. for 3minutes, 35 cycles of (95° C., 1 minute; 63° C. for 30 seconds; 72° C.for 1 minute), followed by 10 minutes at 72° C. The thermocycleremployed was the Amplitron II Thermal Cycler (Thermolyne). Theapproximate 600 bp PCR product was digested with SalI and BamHI, gelpurified and cloned into similarly digested plasmid pUC19 (commerciallyavailable from New England BioLabs, Beverly, Mass.). The ligationmixture was transformed into E. coli strain DH5alpha and transformantsselected on LB plates containing ampicillin. Several colonies were grownovernight in LB media and plasmid DNA isolated using miniplasmid DNAisolation kits purchased from Qiagen, Inc (Valencia, Calif.). Clone LB6was determined to have the correct DNA sequence.

[0049] For expression in E. coli, clone LB6 was digested with NdeI andEcoRI, the approximate 600 bp fragment gel-purified, and cloned intoplasmid pCYB1 (commercially available from New England BioLabs, Beverly,Mass.) that had been digested with the same enzymes and phosphatased.The ligation mixture was transformed into E. coli DH5alpha andtransformants selected on LB ampicillin plates. Plasmid DNA was isolatedfrom several transformants and screened by digestion with NdeI andEcoRI. A correct clone was identified and named pCYB1: wtGH (pBBT120).This plasmid was transformed into E. coli strains JM109 or W3110(available from New England BioLabs and the American Type CultureCollection).

[0050] 2. Construction of STII-GH

[0051] Wild type GH clone LB6 (pUC19: wild type GH) was used as thetemplate to construct a GH clone containing the E. coli STII signalsequence (Picken et al. 1983). Because of its length, the STII sequencewas added in two sequential PCR reactions. The first reaction usedforward primer BB12 and reverse primer BB10. BB10 has the sequence: (SEQID NO: 28) 5′CGCGGATCCGATTAGAATCCACAGCTCCCCTC3′.

[0052] BB12 has the sequence:5′ATCTATGTTCGTTTTCTCTATCGCTACCAACGCTTACGCATTCCCAACCATTCCCTTATCCAG-3′.(SEQ ID NO: 30)

[0053] The PCR reactions were as described for amplifying wild type GHexcept that approximately 4 ng of plasmid LB6 was used as the templaterather than single-stranded cDNA and the PCR conditions were 96° C. for3 minutes, 30 cycles of (95° C. for 1 minute; 63° C. for 30 seconds; 72°C. for 1 minute) followed by 72° C. for 10 minutes. The approximate 630bp PCR product was gel-purified using the Qiaex II Gel Extraction Kit(Qiagen, Inc), diluted 50-fold in water and 2 microliters used astemplate for the second PCR reaction. The second PCR reaction usedreverse primer BB10 and forward primer BB11. BB11 has the sequence:5′CCCCCTCTAGACATATGAAGAAGAACATCGCATTCCTGCTGGCATCTATGTTCGTTTTCTCTATCG-3′.(SEQ ID NO: 29)

[0054] Primer BB11 contains XbaI and NdeI sites for cloning purposes.PCR conditions were as described for the first reaction. The approximate660 bp PCR product was digested with XbaI and BamHI, gel-purified andcloned into similarly cut plasmid pCDNA3.1 (+) (Invitrogen, Inc.Carlsbad, Calif.). Clone pCDNA3.1(+)::stII-GH(5C) or “5C” was determinedto have the correct DNA sequence.

[0055] Clone “5C” was cleaved with NdeI and BarmHI and cloned intosimilarly cut pBBT108 (a derivative of pUC19 which lacks a Pst I site,this plasmid is described below). A clone with the correct insert wasidentified following digestion with these enzymes. This clone,designated pBBT111, was digested with NdeI and SalI, the 660 bp fragmentcontaining the stII-GH fusion gene, was gel-purified and closed into theplasmid expression vector pCYB1 (New England BioLabs) that had beendigested with the same enzymes and phosphatased. A recombinant plasmidcontaining the stII-GH insertion was identified by restrictionendonuclease digestions. One such isolate was chosen for further studiesand was designated pBBT114. This plasmid was transformed into E. colistrains JM109 or W3110 (available from New England BioLabs and theAmerican Type Culture Collection).

[0056] 3. Construction of ompA-GH

[0057] Wild type GH clone LB6 (pUC19: wild type GH) was used as thetemplate to construct a GH clone containing the E. coli ompA signalsequence (Movva et al 1980). Because of its length, the ompA sequencewas added in two sequential PCR reactions. The first reaction usedforward primer BB7:5′GCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCTTCCCAACCATTCCCTTATCCAG3′, (SEQID NO: 31)

[0058] and reverse primer BB10: (SEQ ID NO: 28)5′CGCGGATCCGATTAGAATCCACAGCTCCCCTC3′.

[0059] The PCR reactions were as described for amplifying wild type GHexcept that approximately 4 ng of plasmid LB6 was used as the templaterather than single-stranded cDNA and the PCR conditions were 96° C. for3 minutes, 30 cycles of (95° C. for 1 minute; 63° C. for 30 seconds; 72°C. for 1 minute) followed by 72° C. for 10 minutes. The approximate 630bp PCR product was gel-purified using the Qiaex II Gel Extraction Kit(Qiagen, Inc), diluted 50-fold in water and 2 microliters used astemplate for the second PCR reaction. The second PCR reaction usedreverse primer BB10 and forward PrimerBB6:5′CCCCGTCGACACATATGAAGAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTC3′. (SEQ IDNO: 32)

[0060] PCR conditions were as described for the first reaction. Theapproximate 660 bp PCR product was gel-purified, digested with Sal I andBam H1 and cloned into pUC19 (New England BioLabs) which was cut withSal I and Bam H1 or pCDNA3.1(+) (Invitrogen) which had been cut by Xho Iand Bam H1 ( Sal I and Xho I produce compatible single-strandedoverhangs). When several clones were sequenced, it was discovered thatall pUC19 clones (8/8) contained errors in the region of the ompAsequence. Only one pCDNA3.1(+) clone was sequenced and it contained asequence ambiguity in the ompA region. In order to generate a correctompA-GH fusion gene segments of two sequenced clones which containeddifferent errors separated by a convenient restriction site wererecombined and cloned into the pUC19-derivative that lacks the Pst Isite (see pBBT108 described below). The resulting plasmid, termedpBBT112, carries the ompA-GH fusion gene cloned as an Nde I-Bam H1fragment into these same sites in pBBT108. This plasmid is designatedpBBT112 and is used in PCR-based, site-specific mutagenesis of GH asdescribed below.

[0061] 4. Construction of Pst-pUC19

[0062] To facilitate mutagenesis of the cloned GH gene for constructionof selected cysteine substitution and insertion mutations a derivativeof the plasmid pUC19 (New England BioLabs) lacking a Pst I site wasconstructed as follows. pUC19 plasmid DNA was digested with Pst I andsubsequently treated at 75 deg. C. with PFU DNA Polymerase (Stratagene)using the vendor-supplied reaction buffer supplemented with 200 uMdNTPs. Under these conditions the polymerase will digest the 3′single-stranded overhang created by Pst I digestion but will not digestinto the double-stranded region. The net result will be the deletion ofthe 4 single-stranded bases which comprise the middle four bases of thePst I recognition site. The resulting molecule has double-stranded,i.e., “blunt”, ends. Following these enzymatic reactions, the linearmonomer was gel-purified using the Qiaex II Gel Extraction Kit (Qiagen,Inc). This purified DNA was treated with T4 DNA Ligase (New EnglandBioLabs) according to the vendor protocols, digested with Pst I, andused to transform E. coli DH5alpha. Transformants were picked andanalyzed by restriction digestion with Pst I and Bam H1. One of thetransformants which was not cleaved by Pst I but was cleaved at thenearby Bam H1 site was picked and designated pBBT108.

[0063] 5. Construction of GH Muteins

[0064] GH muteins were generally constructed using site-directedPCR-based mutagenesis as described in PCR Protocols: Current Methods andApplications edited by B. A. White, 1993 Humana Press, Inc., Totowa,N.J. and PCR Protocols: A Guide to Methods and Applications edited byInnis, M. A. et al 1990 Academic Press Inc San Diego, Calif. TypicallyPCR primer oligonucleotides are designed to incorporate nucleotidechanges to the coding sequence of GH that result in substitution of acysteine residue for an amino acid at a specific position within theprotein. Such mutagenic oligonucleotide primers can also be designed toincorporate an additional cysteine residue at the carboxy terminus oramino terminus of the coding sequence of GH. In this latter case one ormore additional amino acid residues could also be incorporated at theamino terminal and/or carboxy terminal to the added cysteine residue ifthat were desirable. Moreover, oligonucleotides can be designed toincorporate cysteine residues as insertion mutations at specificpositions within the GH coding sequence if that were desirable. Again,one or more additional amino acids could be inserted along with thecysteine residue and these amino acids could be positioned at the aminoterminal and/or carboxy terminal to the cysteine residue.

[0065] The cysteine substitution mutation T135C was constructed asfollows. The mutagenic reverse oligonucleotide BB28: (SEQ ID NO: 33)5′CTGCTTGAAGATCTGCCCACACCGGGGGCTGCCATC3′

[0066] was designed to change the codon ACT for threonine at amino acidresidue 135 to a TGT codon encoding cysteine and to span the nearby BglII site. This oligonucleotide was used in PCR along with the forwardoligonucleotide BB34 5′GTAGCGCAGGCCTTCCCAACCATT3′ (SEQ ID NO: 34) whichanneals to the junction region of the ompA-GH fusion gene and is notmutagenic. The PCR was performed in a 50 ul reaction in 1× PCR buffer(Perkin-Elmer buffer containing 1.5 mM MgCl₂), 200 micromolarconcentration of each of the four nucleotides dA, dC, dG and dT, witheach oligonucleotide primer present at 0.5 μM, 5 pg of pBBT112(described above) as template and 1.25 units of Amplitac DNA Polymerase(Perkin-Elmer) and 0.125 units of PFU DNA Polymerase (Stratagene).Reactions were performed in a Robocycler Gradient 96 thermal cycler(Stratagene). The program used entailed: 95 deg C. for 3 minutesfollowed by 25 cycles of 95 deg C. for 60 seconds, 45 deg C. or 50 degC. or 55 deg C. for 75 seconds, 72 deg C. for 60 seconds followed by ahold at 6 deg C. The PCR reactions were analyzed by agarose gelelectrophoresis to identify annealing temperatures that gave significantproduct of the expected size; ˜430 bp. The 45-deg C. reaction was“cleaned up” using the QIAquick PCR Purification Kit (Qiagen), digestedwith Bgl II and Pst I. The resulting 278 bp BE II-Pst I fragment, whichincludes the putative T135C mutation, was gel-purified and ligated intopBBT111 the pUC19 derivative carrying the stII-GH fusion gene (describedabove) which had been digested with Bgl II and Pst I and gel-purified.Transformants from this ligation were initially screened by digestionwith Bgl II and Pst I and subsequently one clone was sequenced toconfirm the presence of the T135C mutation and the absence of anyadditional mutations that could potentially be introduced by the PCRreaction or by the synthetic oligonucleotides. The sequenced clone wasfound to have the correct sequence.

[0067] The substitution mutation S132C was constructed using theprotocol described above for T135C with the following differences:mutagenic reverse oligonucleotide BB295′CTGCTTGAAGATCTGCCCAGTCCGGGGGCAGCCATCTTC3′ (SEQ ID NO: 35) was usedinstead of BB28 and the PCR reaction with annealing temperature of 50deg C. was used for cloning. One of two clones sequenced was found tohave the correct sequence.

[0068] The substitution mutation T148C was constructed using ananalogous protocol but employing a different cloning strategy. Themutagenic forward oligonucleotide BB305′GGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACTGCAACTCACACAAC3′ (SEQ ID NO: 36)was used in PCR with the non-mutagenic reverse primer BB335′CGCGGTACCCGGGATCCGATTAGAATCCACAGCT3′ (SEQ ID NO: 37) which anneals tothe most 3′ end of the GH coding sequence and spans the Bam H1 siteimmediately downstream. PCR was performed as described above with theexception that the annealing temperatures used were 46, 51 and 56 deg C.Following PCR and gel analysis as described above the 46 and 51 deg C.reactions were pooled for cloning. These were digested with Bam H1 andBgl II, gel-purified and cloned into pBBT111 which had been digestedwith Bam H1 and Bgl II, treated with Calf intestinal AlkalinePhosphatase (Promega) according to the vendor protocols, andgel-purified. Transformants from this ligation were analyzed bydigestion with Bam H1 and Bgl II to identify clones in which the 188 bpBam H1-Bgl II mutagenic PCR fragment was cloned in the properorientation. Because Bam H1 and Bgl II generate compatible ends, thiscloning step is not orientation specific. Five of six clones tested wereshown to be correctly oriented. One of these was sequenced and was shownto contain the desired T148C mutation. The sequence of the remainder ofthe 188 bp Bam H1-Bgl II mutagenic PCR fragment in this clone wasconfirmed as correct.

[0069] The construction of the substitution mutation S144C was identicalto the construction of T148C with the following exceptions. Mutagenicforward oligonucleotide BB31 5′GGGCAGATCTTCAAGCAGACCTACTGCAAGTTCGAC3′(SEQ ID NO: 38) was used instead of BB30. Two of six clones tested wereshown to be correctly oriented. One of these was sequenced and was shownto contain the desired S144C mutation. The sequence of the remainder ofthe 188 bp Bam H1-Bgl II mutagenic PCR fragment in this clone wasconfirmed as correct.

[0070] A mutation was also constructed that added a cysteine residue tothe natural carboxy terminus of GH. The construction of this mutation,termed stp192C, was similar to that of T148C, but employed differentoligonucleotide primers. The reverse mutagenic oligonucleotide BB325′CGCGGTACCGGATCCTTAGCAGAAGCCACAGCTGCCCTCCAC3′ (SEQ ID NO: 39) whichinserts a TGC codon for cysteine between the codon for the carboxyterminal phe residue of GH and the TAA translational stop codon andspans the nearby Bam H1 site was used along with BB345′GTAGCGCAGGCCTTCCCAACCATT3′ (SEQ ID NO: 40) which is described above.Following PCR and gel analysis as described above, the 46 deg C.reaction was used for cloning. Three of six clones tested were shown tobe correctly oriented. One of these was sequenced and was shown tocontain the desired stp192C mutation. The sequence of the remainder ofthe 188 bp Bam H1-Bal II mutagenic PCR fragment in this clone wasconfirmed as correct.

[0071] Analogous PCR mutagenesis procedures can be used to generateother cysteine mutations. The choice of sequences for mutagenicoligonucleotides will be dictated by the position where the desiredcysteine residue is to be placed and the propinquity of usefulrestriction endonuclease sites. Generally, it is desirable to place themutation, i.e., the mismatched segment near the middle of theoligonucleotide to enhance the annealing of the oligonucleotide to thetemplate. Appropriate annealing temperatures for any oligonucleotide canbe determined empirically. It is also desirable for the mutagenicoligonucleotide to span a unique restriction site so that the PCRproduct can be cleaved to generate a fragment that can be readily clonedinto a suitable vector, e.g., one that can be used to express the muteinor provides convenient restriction sites for excising the mutated geneand readily cloning it into such an expression vector. Sometimesmutation sites and restriction sites are separated by distances that aregreater than that which is desirable for synthesis of syntheticoligonucleotides: it is generally desirable to keep sucholigonucleotides under 80 bases in length and lengths of 30-40 bases aremore preferable.

[0072] In instances where this is not possible, genes targeted formutagenesis could be re-engineered or re-synthesized to incorporaterestriction sites at appropriate positions. Alternatively, variations ofPCR mutagenesis protocols employed above, such as the so-called“Megaprimer Method” (Barik, S., pp. 277-286 in Methods in MolecularBiology, Vol. 15: PCR Protocols: Current Methods and Applications editedby B. A. White, 1993, Humana Press, Inc., Totowa, N.J.) or “GeneSplicing by Overlap Extension” (Horton, R. Mi., pp. 251-261, in Methodsin Molecular Biology, Vol. 15: PCR Protocols: Current Methods andApplications, edited by B. A. White, 1993, Humana Press, Inc., Totowa,N.J.) can also be employed to construct such mutations.

[0073] 6. Expression of GH in pCYB1

[0074] To express GH in E. coli, pBBT120 (GH gene with no leadersequence cloned into the tac expression vector pCYB1) and pBBT114 (GHgene with stII leader sequence cloned into the tac expression vectorpCYB1) were transformed into E. coli strains JM109 and W3 110. Theparental vector pCYB1 was also transformed into JM109 and W3110. Thesestrains were given the following designations:

[0075] BOB119: JM109 (pCYB1)

[0076] BOB130: W3110 (pCYB1)

[0077] BOB129: JM109 (pBBT120)

[0078] BOB133: W3110 (pBBT120)

[0079] BOB121: JM109 (pBBT114)

[0080] BOB132: W3110 (pBBT114)

[0081] For expression, strains were grown overnight at 37° C. in LuriaBroth (LB) (Sambrook, et al 1989) containing 100 μg/ml ampicillin. Thesesaturated overnight cultures were diluted to ˜0.03 OD at A₆₀₀ in LBcontaining 100 μg/ml ampicillin and incubated at 37° C. in shake flasksin rotary shaker, typically at 250-300 rpm. ODs were monitored and IPTGwas added to a final concentration of 0.5 mM when culture ODs reached˜0.25 −0.5, typically between 0.3 and 0.4. Cultures were sampledtypically at 1, 3, 5 and ˜16 h post-induction. The “˜16 h” time pointsrepresented overnight incubation of the cultures and exact times variedfrom ˜15-20 h. Samples of induced and uninduced cultures were pelletedby centrifugation, resuspended in 1× sample buffer (50 mM Tris-HCl (pH6.8), 2% sodium lauryl sulfate, 10% glycerol, 0.1% bromphenol blue) withthe addition of 1% β-mercaptoethanol when desirable. Samples were boiledfor ˜10 minutes or heated to 95° C. for ˜10 minutes. Samples were cooledto room temperature before being loaded onto SDS polyacrylamide gels orwere stored at −20° C. if not run immediately. Samples were run onprecast 15% polyacrylamide “Ready Gels” (Bio-Rad, Hercules Calif.) usinga Ready Gel Cell electrophoresis apparatus (Bio-Rad) according to thevendor protocols. Typically gels were run at 200 volts for ˜35-45minutes. Gels were stained with Coomassie Blue or were analyzed byWestern Blot following electro-blotting. Coomassie staining of wholecell lysates from strains BOB 129, BOB 133, BOB 121 and BOB 132 showed aband of ˜22 kD that co-migrated with purified recombinant human GHstandard purchased from Research Diagnostics Inc (Flanders, N.J.). Thatband was most prominent in induced cultures following overnightinduction. However, a band was also observed at that molecular weight inuninduced cultures of these same strains and could also be observed withand without induction in the BOB 119 and BOB 130 control strains thatcarried the expression vector pCYB1 lacking the GH gene. To clarify thisobservation, Western Blot analyses were performed on whole cell lysatesof induced cultures of strains BOB119, BOB130, BOB129, BOB133, BOB121,and BOB132. Western blots were performed with polyclonal rabbitanti-human GH antiserum purchased from United States Biological;catalogue # G 9000-11 (Swampscott, Mass.) This primary antibody was usedat a 1:5000 dilution and its binding was detected with goat anti-rabbitIgG Fc conjugated to alkaline phosphatase, (product # 31341) purchasedfrom Pierce (Rockford, Ill.). This secondary antibody was used at a1:10,0000 dilution. Alkaline phosphatase activity was detected using theImmunoPure® Fast Red TR/AS-MX Substrate Kit (Pierce, Rockford Ill.)according to the vendor protocols. The Western Blots clearlydemonstrated presence of GH in lysates of induced cultures of BOB129,BOB133, BOB121, and BOB132 at both 3 and 16 h post-induction. In theinduced culture of control strains, BOB119 and BOB130, no GH wasdetected by Western blot at 3 or 16 h post-induction time points.

[0082] In these preliminary experiments, the highest yields of GH wereobtained from BOB132 W3110(pBBT114) in which the GH gene is fuseddownstream of the stII secretion signal sequence. This strain was testedfurther to determine if the GH protein was secreted to the periplasm aswould be expected. An induced culture of BOB132 was prepared asdescribed above and subjected to osmotic shock according to theprocedure of Koshland and Botstein (Cell 20 (1980) pp. 749-760). Thisprocedure ruptures the outer membrane and releases the contents of theperiplasm into the surrounding medium. Subsequent centrifugationseparates the periplasm contents, present in the supernatant from theremainder of the cell-associated components. In this experiment, thebulk of the GH synthesized by BOB132 was found to be localized to theperiplasm. This result is consistent with the finding that the bulk ofthe total GH is also indistinguishable in size from the purified GHstandard, which indicated that the stII signal sequence had beenremoved. This is indicative of secretion. A larger scale (500 ml)culture of BOB132 was also induced, cultured overnight and subjected toosmotic shock according to the procedure described by Hsiung et al, 1986(Bio/Technology 4, pp. 991-995). Gel analysis again demonstrated thatthe bulk of the GH produced was soluble, periplasmic, andindistinguishable in size from the GH standard. This material could alsobe quantitatively bound to, and eluted from, a Q-Sepharose column usingconditions very similar to those described for recombinant human GH byBecker and Hsiung, 1986 (FEBS Lett 204 pp145-150).

[0083] 7. Cloning the Human GH Receptor

[0084] The human GH receptor was cloned by PCR using forward primer BB3and reverse primer BB4. BB3 has the sequence: (SEQ ID NO: 26)5′-CCCCGGATCCGCCACCATGGATCTCTGGCAGCTGCTGTT-3′.

[0085] BB4 has the sequence: (SEQ ID NO: 27)5′CCCCGTCGACTCTAGAGCTATTAAATACGTAGCTCTTGGG-3′.

[0086] The template was single-stranded cDNA prepared from human liver(commercially available from CLONTECH Laboratories). Primers BB3 and BB4contain BamHI and SalI restriction sites, respectively, for cloningpurposes. The 100 μl PCR reactions contained 2.5 ng of thesingle-stranded cDNA and 20 picomoles of each primer in 1× PCR buffer(Perkin-Elmer buffer containing MgCl₂), 200 micromolar concentration ofeach of the four nucleotides dA, dC, dG and dT, 2.5 units of Taqpolymerase (Perkin-Elmer) and 2.5 units of Pfu polymerase (Stratagene,Inc). The PCR reaction conditions were: 96° C. for 3 minutes, 35 cyclesof (95° C., 1 minute; 58° C. for 30 seconds; 72° C. for 2 minutes),followed by 10 minutes at 72° C. The thermocycler employed was theAmplitron II Thermal Cycler (Thermolyne). The approximate 1.9 kb PCRproduct was digested with BamHI and SalI and ligated with similarly cutplasmid pUC19 ( New England BioLabs). However, none of the transformantsobtained from this ligation reaction contained the 1.9 kb PCR fragment.Leung et al (Nature 1987 330 pp537-543) also failed to obtainfull-length cDNA clones of the human GH receptor in pUC19. Subsequentlythe PCR fragment was cloned into a low copy number vector, pACYC184 (NewEngland BioLabs) at the BamHI and SalI sites in this vector. Such cloneswere obtained at reasonable frequencies but E coli strains carrying thecloned PCR fragment grew poorly, forming tiny and heterogeneous lookingcolonies, in the presence of chloramphenicol, which is used to selectfor maintenance of pACYC 184.

[0087] The PCR fragment was simultaneously cloned into pCDNA3.1 (+)(Invitrogen). The approximate 1.9 kb PCR product was digested with BamHIand SalI and ligated into the BamHI and XhoI cloning sites of pCDNA3.1(+). Only infrequent transformants from this ligation contained thecloned GH receptor cDNA and all of those were found to contain deletionsof segments of the receptor coding sequence. One of these clones wassequenced and found to contain a deletion of 135 bp within the GHreceptor coding sequence: the sequence of the rest of the gene was inagreement with that reported by Leung et at (1987).

[0088] 8. Cloning the Rabbit GH Receptor

[0089] The rabbit GH receptor was cloned by PCR using forward primer BB3(described above) and reverse primer BB36. BB36 has the sequence (SEQ IDNO: 41) 5′CCCCGTCGACTCTAGAGCCATTAGATACAAAGCTCTTGGG3′

[0090] and contains XbaI and Sal I restriction sites for cloningpurposes. Rabbit liver poly(A)⁺ mRNA was purchased from CLONTECH, Inc.and used as the substrate in first strand synthesis of single-strandedcDNA to produce template for PCR amplification. First strand synthesisof single-stranded cDNA was accomplished using a 1 st Strand cDNASynthesis Kit for RT-PCR (AMV) kit from Boehringer Mannheim Corp(Indianapolis, Ind.) according to the vendor protocols. Parallel firststrand cDNA syntheses were performed using random hexamers or BB36 asthe primer. Subsequent PCR reactions with the products of the firststrand syntheses as templates, were carried out with primers BB3 andBB36 according to the 1 st Strand cDNA Synthesis Kit for RT-PCR (AMV)kit protocol and using 2.5 units of Amplitac DNA Polymerase(Perkin-Elmer) and 0.625 units of Pfu DNA Polymerase (Stratagene). ThePCR reaction conditions were 96° C. for 3 minutes, 35 cycles of (95° C.,1 minute; 58° C. for 30 seconds; 72° C. for 2 minutes), followed by 10minutes at 72° C. The thermocycler employed was the Amplitron II ThermalCycler (Thermolyne). The expected 1.9 kb PCR product was observed in PCRreactions using random hexamer-primed or BB36 primed cDNA as template.The random hexamer-primed cDNA was used in subsequent cloningexperiments. It was digested with Bam H1 and XbaI and run out over a1.2% agarose gel. This digest generates two fragments (˜365 bp and ˜1600bp) because the rabbit GH receptor gene contains an internal Bam H1site. Both fragments were gel-purified. Initially the ˜1600 bp BamH1-XbaI fragment was cloned into pCDNA3.1(+) which had been digestedwith these same two enzymes. These clones were readily obtained atreasonable frequencies and showed no evidence of deletions as determinedby restriction digests and subsequent sequencing. To generate a fulllength clone, one of the plasmids containing the 1600 bp Bam H1-Xba Ifragment (pCDNA3.1(+):: rab-ghr-2A) was digested with Bam H1, treatedwith Calf Intestinal Alkaline Phosphatase (Promega) according to thevendor protocols, gel-purified and ligated with the gel purified ˜365 bpBam H1 fragment that contains the 5′ portion of the rabbit GH receptorgene. Transformants from this ligation were picked and analyzed byrestriction digestion and PCR to confirm the presence of the ˜365 bpfragment and to determine its orientation relative to the distal segmentof the rabbit GH receptor gene. Three out of four clones analyzed werefound to contain the ˜365 bp fragment cloned in the correct orientationfor reconstitution of the rabbit GH receptor gene. The lack ofcomplications in the cloning in E coli of the rabbit gene, in contrastto the human gene, is consistent with the results of Leung et al (1987)who also readily obtained full length cDNA clones for the rabbit GHreceptor gene but were unable to clone a full length cDNA of the humangene in E coli. The rabbit GH receptor can be employed in assays withhuman GH as a ligand as it has been shown that the human GH binds therabbit receptor with high affinity (Leung et al 1987). Plasmidscontaining the cloned rabbit GH receptor should be sequenced to identifya rabbit GH receptor cDNA with the correct sequence before use.

[0091] 9. Construction of a Human/Rabbit Chimeric GH Receptor Gene

[0092] As an alternative to the rabbit receptor, a chimeric receptorcould be constructed which combines the extracellular domain of thehuman receptor with the transmembrane and cytoplasmic domains of therabbit receptor. Such a chimeric receptor could be constructed byrecombining the human and rabbit genes at the unique Nco I site that ispresent in each (Leung et al 1987). Such a recombinant, containing thehuman gene segment located 5′ to, or “upstream” of the Nco I site andthe rabbit gene segment 3′ to, or “downstream” of the Nco I site wouldencode a chimeric receptor of precisely the desired type, having theextracellular domain of the human receptor with the transmembrane andcytoplasmic domains of the rabbit receptor. This would allow analysis ofthe interaction of GH, GH muteins, and PEGylated GH muteins with thenatural receptor binding site but could avoid the necessity of cloningthe full length human GH receptor in E coli.

[0093] The GH muteins can be expressed in a variety of expressionsystems such as bacteria, yeast or insect cells. Vectors for expressingGH muteins in these systems are available commercially from a number ofsuppliers such as Novagen, Inc. (pET15b for expression in E. coli), NewEngland Biolabs (pC4B1 for expression in E. coli) Invitrogen (pVL1392,pVL1 393, and pMELBAC for expression in insect cells using Baculovirusvectors, Pichia vectors for expression in yeast cells, and pCDNA3 forexpression in mammalian cells). Gn has been successfully produced in E.coli as a cytoplasmic protein and as a secreted, periplasmic, proteinusing the E. coli OmpA or STII signal sequences to promote secretion ofthe protein into the periplasmic space (Chang et al., 1987; Hsiung etal., 1986). It is preferable that the GH muteins are expressed assecreted proteins so that they do not contain an N-terminal methionineresidue, which is not present in the natural human protein. Forexpression in E. coli, DNA sequences encoding GH or GH muteins can becloned into E. coli expression vectors such as pET15b that uses thestrong T7 promoter or pCYB1 that uses the TAC promoter. Adding IPTG(isopropylthiogalactopyranoside, available form Sigma Chemical Company)to the growth media, can induce expression of the protein. RecombinantGH will be secreted into the periplasmic space from which it can bereleased by, and subsequently purified, following osmotic shock (Beckerand Hsiung, 1986). The protein can be purified further using otherchromatographic methods such as ion-exchange, hydrophobic interaction,size-exclusion and reversed phase chromatography, all of which are wellknown to those of skill in the art (e.g., see Becker and Hsiung, 1986).Protein concentrations can be determined using commercially availableprotein assay kits such as those sold by BioRad Laboratories (Richmond,Calif.). If the GH proteins are insoluble when expressed in E. coli theycan be refolded using procedures well known to those skilled in the art(see Cox et al., 1994 and World patent applications WO9422466 andWO9412219).

[0094] Alternatively, the proteins can be expressed in insect cells assecreted proteins. The expression plasmid can be modified to contain theGH signal sequence to promote secretion of the protein into the medium.The cDNAs can be cloned into commercially available vectors, e.g.,pVL1392 from Invitrogen, Inc., and used to infect insect cells. The GHand GH muteins can be purified from conditioned media using conventionalchromatography procedures. Antibodies to rhGH can be used in conjunctionwith Western blots to localize fractions containing the GH proteinsduring chromatography. Alternatively, fractions containing GH can beidentified using ELISA assays.

[0095] The cysteine-added GH variants also can be expressed asintracellular or secreted proteins in eukaryotic cells such as yeast,insect cells or mammalian cells. Vectors for expressing the proteins andmethods for performing such experiments are described in catalogues fromvarious commercial supply companies such as Invitrogen, Inc.,Stratagene, Inc. and ClonTech, Inc. The GH and GH muteins can bepurified using conventional chromatography procedures.

[0096] Biological activity of the GH muteins can be measured using acell line that proliferates in response to GH. Fuh et al. (1992) createda GH-responsive cell line by stably transforming a myeloid leukemia cellline, FDC-P1, with a chimeric receptor comprising the extracellulardomain of the rabbit GH receptor fused to the mouse G-CSF receptor. Thiscell line proliferates in response to GH with a half maximal effectiveconcentration (EC₅₀) of 20 picomolar. A similar cell line can beconstructed using the published sequences of these receptors andstandard molecular biology techniques (Fuh et al., 1992). Alternatively,the extracellular domain of the human GH receptor can be fused to themouse G-CSF receptor using the published sequences of these receptorsand standard molecular biology techniques. Transformed cells expressingthe chimeric receptor can be identified by flow cytometry using labeledGH, by the ability of transformed cells to bind radiolabeled GH, or bythe ability of transformed cells to proliferate in response to added GH.Purified GH and GH muteins can be tested in cell proliferation assaysusing cells expressing the chimeric receptor to measure specificactivities of the proteins. Cells can be plated in 96-well dishes withvarious concentrations of GH or GH muteins. After 18 h, cells aretreated for 4 hours with ³H-thymidine and harvested for determination ofincorporated radioactivity. The EC₅₀ can be determined for each mutein.Assays should be performed at least three times for each mutein usingtriplicate wells for each data point. GH muteins displaying similaroptimal levels of stimulation and EC₅₀ values comparable to or greaterthan wild type GH are preferable.

[0097] GH muteins that retain in vitro activity can be PEGylated using acysteine-reactive 8 kDa PEG-maleimide (or PEG-vinylsulfone) commerciallyavailable from Shearwater, Inc. Generally, methods for PEGylating theproteins with these reagents will be similar to those described in worldpatent applications WO 9412219 and WO 9422466 and PCT applicationUS95/06540, with minor modifications. The recombinant proteins must bepartially reduced with dithiothreitol (DTT) in order to achieve optimalPEGylation of the free cysteine. Although the free cysteine is notinvolved in a disulfide bond, it is relatively unreactive tocysteine-reactive PEGs unless this partial reduction step is performed.The amount of DTT required to partially reduce each mutein can bedetermined empirically, using a range of DTT concentrations. Typically,a 5-10 fold molar excess of DTT for 30 min at room temperature issufficient. Partial reduction can be detected by a slight shift in theelution profile of the protein from a reversed-phase column. Care mustbe taken not to over-reduce the protein and expose additional cysteineresidues. Over-reduction can be detected by reversed phase-HPLC (theprotein will have a retention time similar to the fully reduced anddenatured protein) and by the appearance of GH molecules containing twoPEGs (detectable by a molecular weight change on SDS-PAGE). Wild type GHcan serve as a control since it should not PEGylate under similarconditions. Excess DTT can be removed by size exclusion chromatographyusing spin columns. The partially reduced protein can be reacted withvarious concentrations of PEG-maleimide (PEG: protein molar ratios of1:1, 5:1,10:1 and 50:1) to determine the optimum ratio of the tworeagents. PEGylation of the protein can be monitored by a molecularweight shift using sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). The lowest amount of PEG that givessignificant quantities of mono-pegylated product without givingdi-pegylated product will be considered optimum (80% conversion tomono-pegylated product is considered good). Generally, mono-PEGylatedprotein can be purified from non-PEGylated protein and unreacted PEG bysize-exclusion or ion exchange chromatography. The purified PEGylatedprotein can be tested in the cell proliferation assay described above todetermine its specific activity.

[0098] The above experiments will allow identification of amino acids inthe B-C loop, C-D loop or N-terminal end of the A-B loop in GH that canbe changed to cysteine residues, PEGylated and retain in vitrobiological activity. These muteins can be tested in animal diseasemodels well known in the art.

[0099] Experiments can be performed to confirm that the PEG molecule isattached to the protein at the proper site. This can be accomplished byproteolytic digestion of the protein, purification of the PEG peptide(which will have a large molecular weight) by size exclusion, ionexchange or reversed phase chromatography, followed by amino acidsequencing or mass spectroscopy. The PEG-coupled amino acid will appearas a blank in the amino acid sequencing run.

[0100] The pharmacokinetic properties of the PEG-GH proteins can bedetermined as follows, or as described in world patent applicationWO9422466. Pairs of rats or mice can receive an intravenous bolusinjection of the test proteins. Circulating levels of the proteins aremeasured over the course of 24 h by removing a small sample of bloodfrom the animals at desired time points. Circulating levels of the testproteins can be quantitated using ELISA assays. Additional experimentscan be performed using the subcutaneous route to administer theproteins. Similar experiments should be performed with the non-PEGylatedprotein to serve as a control. These experiments will reveal whetherattachment of a PEG reagent to the protein alters its pharmacokineticproperties. Covalent modification of the protein with PEG shouldincrease the protein's circulating half-life relative to the unPEGylatedprotein. Larger PEG molecules and/or attachment of multiple PEGmolecules should lengthen the circulating half-life longer than smallerPEG molecules.

[0101] PEG-GH proteins can be tested in rodent models of growth hormonedeficiency (Cox et al., 1994) and cachexia (Tomas et al., 1992; Read etal., 1992) to determine optimum dosing schedules and demonstrateefficacy. These studies can explore different size PEG molecules, e.g.,8 and 20 kDa, and dosing schedules to determine the optimum PEG size anddosing schedule. It is expected that the larger PEG molecule willincrease the circulating half-life greater than the smaller PEG moleculeand will require less frequent dosing. However, large proteinspotentially may have reduced volumes of distribution in vivo; thus, itis possible a 20 kDa PEG attached to GH will limit bioavailability,reducing its efficacy. Rodent models will allow determination of whetherthis is the case. Once the optimum dosing schedules and PEG sizes aredetermined, the efficacy of PEG-GH to GH can be compared in the animalmodels. While all PEG-GH proteins having GH activity are included in theinvention, the preferred PEG-GH proteins are those that enhance growthequal or superior to GH, but which can be given less frequently. PEG-GHshould be more efficacious than GH when both are administered using theless frequent dosing schedules.

[0102] One GH deficiency model that can be used is a hypophysectomizedrat. GH stimulates body weight gain and bone and cartilage growth inthis model (Cox et al., 1994). Hypophysectomized rats can be purchasedfrom Charles River. Rats can be injected with GH, PEG-GH or placebo andweight gain measured daily over a 10-14 day period. At time ofsacrifice, tibial epiphysis width can be determined as a measure of bonegrowth. Experimental methods for performing these studies are describedin Cox et al. (1994).

[0103] The efficacy of PEG-GH in rodent cachexia models can be tested ina similar manner. Daily administration of dexamethasone, via osmoticpumps or subcutaneous injection, can be used to induce weight loss(Tomas et al., 1992; Read et al., 1992; PCT patent applicationUS95/06540).

EXAMPLE 2 Cysteine-Added Variants of Erythropoietin

[0104] This example relates to cysteine-added variants of erythropoietin(EPO). EPO is the hormone primarily responsible for stimulatingerythropoiesis or red blood cell formation. EPO acts on immature redblood cell precursors to stimulate their further proliferation anddifferentiation into mature red blood cells. A commercial pharmaceuticalversion is available from Amgen, Inc. Human EPO is a 35-39 kDaglycoprotein secreted by the adult kidney. The mature human proteincontains 166 amino acids and is heavily glycosylated. The sequence ofhuman EPO (SEQ ID NO: 2) is shown in Lin et al 1985 and Jacobs et al.1985, which are incorporated herein by reference. The primary sequenceof EPO is highly conserved among species (greater than 80% identity; Wenet al., 1994). Sugar groups account for greater than 40% of theprotein's mass. Human EPO contains three N-linked glycosylation sitesand one O-linked glycosylation site. The N-linked glycosylation sitesare conserved in different species whereas the O-linked glycosylationsite is not. The extensive glycosylation of EPO has prevented theprotein's crystallization, so the X-ray structure of the protein is notknown. Human EPO contains four cysteine residues. The disulfideassignments are Cys7 to Cys161 and Cys29 to Cys33. Cys33 is notconserved in mouse EPO, suggesting that the Cys29-Cys33 disulfide bondis not critical to mouse EPO's structure or function. This conclusionalso seems to hold for human EPO (Boissel et al., 1993).

[0105] The amino acid sequence of EPO is consistent with the proteinbeing a member of the GH supergene family and mutational studies supportthis view of EPO's structure (Boissel et al., 1993; Wen et al., 1994). Amodel of the three dimensional structure of EPO, modeled after the GHstructure has been proposed (Boissel et al., 1993; Wen et al., 1994).Amino acids in EPO important for receptor binding have been identifiedthrough mutagenesis experiments and reside primarily in the N-terminalhalf of presumptive helix A and the C-terminal half of presumptive helixD (Boissel et al., 1993; Wen et al., 1994; Matthews et al., 1996). Onlya single cell surface receptor for EPO has been identified (D'Andrea etal., 1989). It is believed that EPO dimerizes its receptor in much thesame way that GH dimerizes its receptor (Matthew's et al., 1996).

[0106] Human EPO contains three sites for N-linked glycosylation(asparagine-24,-38 and -83) and one site for O-linked glycosylation(serine-126). The N-linked glycosylation sites are located in the A-Band B-C loops and the O-glycosylation site is located in the C-D loop.The N-linked glycosylation sites are conserved among species whereas theO-linked glycosylation site is adsent in rodent EPO (Wen et al., 1993).A non-O-linked glycosylated human variant containing methionine atposition 126 has been described (U.S. Pat. No. 4,703,008). The N-linkedsugar groups are heavily branched and contain terminal sialic acidresidues (Sasaki et al., 1987; Takeuchi et al., 1988). N-38 and N-83contain the most highly branched oligosaccharides (Sasaki et al., 1988).

[0107] The terminal sialic residues on EPO are critical for theprotein's in vivo function because removal of these residues bydigestion eliminates in vivo activity (Fukada et al., 1989; Spivak andHogans, 1989). Loss of activity correlates with faster clearance of theasialated protein from the body. The circulating half-life of theasialated protein in rats is less than ten minutes, in contrast to thatof the sialated protein, which is approximately 2 hr (Fukada et al.,1989; Spivak and Hogans, 1989). Thus, in vivo activity of EPO directlycorrelates with its circulating half-life.

[0108] The role of N-linked sugars in EPO's biological activities haSbeen better defined by mutating, individually and in combination, thethree asparagine residues comprising the N-linked glycosylation sites.EPO muteins in which only a single N-linked glycosylation site wasmutated, i.e., N24Q, N38Q and N83Q, were secreted from mammalian cellsas efficiently as wild type EPO, indicating that N-linked glycosylationat all three sites is not required for protein secretion (N24Q indicatesthat the asparagine at position 24 is mutated to glutamine). Incontrast, EPO muteins in which two or more N-linked glycosylation siteswere mutated were secreted less efficiently than wild type EPO frommammalian cells (Yamaguchi et al., 1991; Delorme et al., 1992).Mutagenesis studies found that each of the single N-linked glycosylationsite muteins had in vitro biological activities equal to or greater thanwild type EPO. Thus, it was concluded that none of the N-linkedglycosylation sites is essential for secretion or in vitro biologicalactivity of EPO. In fact, removal of one of the glycosylation sitesseemed to improve biological activity (Yamaguchi et al., 1991).

[0109] The in vivo biological activity of N-linked glycosylation muteinswas studied by two groups. Yamaguchi et al. (1991) concluded that theN24Q and N83Q muteins had in vivo activities greater than wild type EPO,which correlated with their increased in vitro activities. These authorsfound that the N38Q mutein had decreased in vivo activity, about 60% ofwild type EPO. N38 is the most heavily branched of the three N-linkedglycosylation sites (Sasaki et al., 1988). Delorme et al. (1992)reported that mutating any of the N-linked glycosylation sites reducedin vivo biological activity by about 50%. Muteins in which two or moreglycosylation sites were mutated had decreased in vivo activities inboth studies.

[0110] The above studies indicate that some N-linked glycosylation isrequired for in vitro and in vivo activity of EPO. Individually,however, none of the three glycosylation sites is absolutely essentialfor activity. The N-linked sugars increase the apparent molecular weightof EPO and prolong its circulating half-life, which correlates withbioactivity. Natural EPO and EPO manufactured in mammalian cells havecomplex N-linked sugars containing galactose and terminal sialic acidresidues. The galactose residues are recognized by specific receptors onhepatocytes and promote rapid clearance of EPO from the body unless thegalactose residues are masked by the terminal sialic acid residues.

[0111] Mutagenesis studies concluded that O-linked glycosylation is notrequired for in vitro or in vivo function of EPO (Delorme et al., 1992).This is in keeping with the observation that rodent EPO is notO-glycosylated and with the existence of a naturally occurring human EPOvariant in which serine-126 is replaced by methionine, with acorresponding lack of O-linked glycosylation. Mutagenesis of serine-126revealed that certain amino acid changes at this site (to valine,histidine or glutamic acid) yielded EPO muteins with biologicalactivities similar to wild type EPO, whereas other amino acid changes(to alanine or glycine) resulted in EPO molecules with severely reducedactivities (Delorme et al., 1992). The effect of changing serine-126 tocysteine was not studied. The in vivo bioactivity of S126V EPO was foundto be similar to wild type EPO (Delorme et al., 1992).

[0112] The requirement for complex, N-linked carbohydrates containingterminal sialic acid residues for in vivo activity of EPO has limitedcommercial manufacture of the protein to mammalian cells. The importantfunctions of the sialated N-linked sugars are to prevent proteinaggregation, increase protein stability and prolong the circulatinghalf-life of the protein. The terminal sialic acid residues prolongEPO's circulating half-life by masking the underlying galactoseresidues, which are recognized by specific receptors on hepatocytes andpromote clearance of the asialated protein. EPO can be produced ininsect cells and is N-glycosylated and fully active in vitro; itsactivity in vivo has not been reported (Wojchowski et al., 1987).

[0113] This example provides for the design of cysteine-added EPOvariants and their use in preparing conjugates using cysteine-reactivePEGs and other cysteine-reactive moieties. Certain amino acids in EPOare non-essential for biological activity and can be mutated to cysteineresidues without altering the normal disuifide binding patter, andoverall conformation of the molecule. These amino acids are located inthe A-B loop (amino acids 23-58 of the mature protein sequence), the B-Cloop (amino acids 77-89 of the mature protein sequence), the C-D loop(amino acids 108-131 of the mature protein sequence), proximal to helixA (amino acids 1-8) and distal to helix D (amino acids 153-166 of themature protein sequence). Also contemplated as preferred sites foradding cysteine residues are at the N-terminus or C-terminus of theprotein sequence. Preferred sites for cysteine substitutions are theO-linked glycosylation site (serine-126) and the amino acids comprisingthe three N-linked glycosylation sites (N24, I25, T26, N38, 139, T40,N83, S84, S85). Glycosylation sites are attractive sites for introducingcysteine substitutions and attaching PEG molecules to EPO because (1)these sites are surface exposed; (2) the natural protein can toleratebulky sugar groups at these positions; (3) the glycosylation sites arelocated in the putative loop regions and away from the receptor bindingsite (Wen et al., 1994); and (4) mutagenesis studies indicate thesesites (at least individually) are not essential for in vitro or in vivoactivity (Yamaguchi et al., 1991; Delorme et al., 1992). As discussedabove, the local conformation of the region encompassing theO-glycosylation site region seems to be important for biologicalactivity. Whether a cysteine substitution at position 126 affectsbiological activity has not been studied. The cysteine-29 to cysteine-33disulfide bond is not necessary for biological activity of EPO becausechanging both residues to tyrosine simultaneously yielded a biologicallyactive EPO protein (Boissel et al., 1993; Wen et al., 1994). A “free”cysteine can be created by changing either cysteine-29 or cysteine-33 toanother amino acid. Preferred amino acid changes would be to serine oralanine. The remaining “free” cysteine (cysteine-29 or cysteine-33)would be a preferred site for covalently modifying the protein withcysteine-reactive moieties.

[0114] Bill et al. (1995) individually substituted cysteine for N24, N38and N83 and reported that the muteins had greatly reduced in vitrobiological activities (less than 20% of wild type activity). Bill etal.(1995) expressed the EPO variants as fusion proteins (fused toglutathionine-S-transferase) in bacteria. One aspect of the presentinvention is to provide expression systems in which the N24C, N38C andN83C EPO variants will have in vitro biological activities more similarto wild type EPO.

[0115] U.S. Pat. No. 4,703,008 contemplates naturally occurring variantsof EPO as well as amino acid substitutions that are present in EPOproteins of mammals. Ovine EPO contains a cysteine residue at position88 of the polypeptide chain. The inventor is unaware of any othernaturally occurring human or animal cysteine variants of EPO in whichthe cysteine residue occurs in the polypeptide regions disclosed hereinas being useful for generating cysteine-added EPO variants. U.S. Pat.No. 4,703,008 specifically teaches away from cysteine-added EPO variantsby suggesting that expression of EPO might be improved by deletingcysteine residues or substituting naturally occurring cysteine residueswith serine or histidine residues.

[0116] The mature protein form of EPO can contain 165 or 166 amino acidsbecause of post-translational removal of the C-terminal arginine.Asp-165 is the C-terminus of the 165 amino acid form and Agr-166 is theC-terminal amino acid of the 166 amino acid form. The cysteinesubstitution and insertion mutations described herein can compriseeither the 165 or 166 amino acid forms of mature EPO.

[0117] A cDNA encoding EPO can be cloned using the polymerase chainreaction (PCR) technique from the human HepG2 or Hep3B cell lines, whichare known to express EPO when treated with hypoxia or cobalt chloride(Wen et al., 1993) and are available from the American Type CultureCollection (ATCC). Cysteine mutations can be introduced into the cDNA bystandard phage, plasmid or PCR mutagenesis procedures as described forGH. As described above, the preferred sites for introduction of cysteinesubstitution mutations are in the A-B loop, the B-C loop, the C-D loopand the region proximal to helix A and distal to helix D. The mostpreferred sites in these regions are the N— and O-linked glycosylationsites: S126C; N24C; I25C, T26C; N38C; I39C, T40C; N83C, S84C and S85C.Other preferred sites for cysteine substitution mutagenesis are in theA-B loop, the B-C loop and the C-D loop, amino acids surrounding theglycosylation sites and the region of the protein proximal to helix Aand distal to helix D (Boissel et al., 1993; Wen et al., 1994). Otherpreferred sites for cysteine substitutions in these regions are: A1, P2,P3, R4, D8, S9, T27, G28, A30, E31, H32, S34, N36, D43, T44, K45, N47,A50, K52, E55, G57, Q58, G77, Q78, A79, Q86, W88, E89, T107, R110, A111,G113, A114, Q115, K116, E117, A118, S120, P121, P122, D123, A124, A125,A127, A128, T132, K154, T157, G158, E159, A160, T163, G164, D165, andR166. Cysteine residues also can be introduced proximal to the firstamino acid of the mature protein, i.e., proximal to A1, or distal to thefinal amino acid in the mature protein, i.e., distal to D165 or R166.Other variants in which cys-29 or cys-33 have been replaced with otheramino acids, preferably serine or alanine, also are provided.

[0118] Wild type EPO and EPO muteins can be expressed using insect cellsto determine whether the cysteine-added muteins are biologically active.DNAs encoding EPO/EPO muteins can be cloned into the Baculovirusexpression vector pVL1392 (available from Invitrogen, Inc. and SigmaCorporation (St. Louis, Mo.) and used to infect insect cells.Recombinant Baculoviruses producing EPO can be identified by Westernblots of infected insect cell conditioned media using polyclonalanti-human EPO antiserum (available from R&D Systems). The secreted EPOmutein proteins can be purified by conventional chromatographicprocedures well known to those of skill in the art. Proteinconcentrations can be determined using commercially available proteinassay kits or ELISA assay kits (available from R&D Systems and Bio-RadLaboratories).

[0119] Purified EPO and EPO muteins can be tested in cell proliferationassays using EPO-responsive cell lines such as UT7-epo (Wen et al.,1994) or TF1 (available from the ATCC) to measure specific activities ofthe proteins. Cells can be plated in 96-well microtiter plates withvarious concentrations of EPO. Assays should be performed in triplicate.After 1-3 days in culture, cell proliferation can be measured by³H-thymidine incorporation, as described above for GH. The concentrationof protein giving half-maximal stimulation (EC₅₀) can be determined foreach mutein. Assays should be performed at least three times for eachmutein, with triplicate wells for each data point. EC₅₀ values can beused to compare the relative potencies of the muteins. Alternatively,cell proliferation in response to added EPO muteins can be analyzedusing an MTT dye-exclusion assay (Komatsu et al. 1991). Proteinsdisplaying similar optimal levels of stimulation and EC₅₀ valuescomparable to or greater than wild type EPO are preferable.

[0120] The above studies confirm identification of amino acid residuesin EPO that can be changed to cysteine residues and retain biologicalactivity. Muteins that retain activity can be PEGylated using acysteine-reactive 8 kDa PEG-maleimide as described above for GH muteins.Wild type EPO should be used as a control since it should not react withthe cysteine-reactive PEG under identical partial reduction conditions.The lowest amount of PEG that gives significant quantities ofmono-PEGylated product without giving di-PEGylated product should beconsidered optimum. Mono-PEGylated protein can be purified fromnon-PEGylated protein and unreacted PEG by size-exclusion or ionexchange chromatography. The purified PEGylated proteins should betested in the cell proliferation assay described above to determinetheir bioactivities.

[0121] One or more of the PEGylated EPO muteins that retain in vitrobioactivity, are candidates for testing in animal disease models.PEGylation of the protein at the proper amino acid can be determined asdescribed for GH.

[0122] In vivo testing of PEGylated EPO muteins expressed using insectcells may require that they be re-engineered for expression in mammaliancells to ensure proper glycosylation. PEG-EPO candidates produced usinginsect cells can be tested in the animal models described below todetermine if they are active in vivo and whether they are as active asPEG-EPO produced using mammalian cell expression systems. For expressionin mammalian cells, the EPO muteins can be subdloned into commerciallyavailable eukaryotic expression vectors and used to stably transformChinese Hamster Ovary (CHO) cells (available from the ATCC). Sublinescan be screened for EPO expression using ELISA assays. Sufficientquantities of the insect cell- and mammalian cell-produced EPO muteinscan be prepared to compare their biological activities in animal anemiamodels.

[0123] In vivo bioactivities of the EPO muteins can be tested using theartificial polycythemia or starved rodent models (Cotes and Bangham.,1961; Goldwasser and Gross., 1975). In the starved rodent model, ratsare deprived of food on day one and treated with test samples on daystwo and three. On day four, rats receive an injection of radioactiveiron-59. Approximately 18 h later, rats are anesthetized and bloodsamples drawn. The percent conversion of labeled iron into red bloodcells is then determined. In the artificial polycythemia model, mice aremaintained in a closed tank and exposed for several days to hypobaricair. The animals are then brought to normal air pressure. Red blood cellformation is suppressed for several days. On day four or six afterreturn to normal air pressure, mice are injected with erythropoietin orsaline. Mice receive one injection per day for one to two days. One daylater the animals receive an intravenous injection of labeled iron-59.The mice are euthanized 20 h later and the amount of labeled ironincorporated into red blood cells determined. EPO stimulates red bloodcell formation in both models as measured by a dose-dependent increasein labeled iron incorporated into red blood cells. In both modelsdifferent dosing regimens and different times of injections can bestudied to determine if PEG-EPO is biologically active and/or morepotent and produces longer acting effects than natural EPO.

EXAMPLE 3 Alpha Interferon

[0124] Alpha interferon is produced by leukocytes and has antiviral,anti-tumor and immunomodulatory effects. There are at least 20 distinctalpha interferon genes that encode proteins that share 70% or greateramino acid identity. Amino acid sequences of the known alpha interferonspecies are given in Blatt et al. (1996). A “consensus” interferon thatincorporates the most common amino acids into a single polypeptide chainhas been described (Blatt et al., 1996). A hybrid alpha interferonprotein may be produced by splicing different parts of alpha interferonproteins into a single protein (Horisberger and Di Marco, 1995). Somealpha interferons contain N-linked glycosylation sites in the regionproximal to helix A and near the B-C loop (Blatt et al. 1966). The alpha2 interferon protein (SEQ ID NO: 3) contains four cysteine residues thatform two disulfide bonds. The cys1-cys98 disulfide bond (cys1-cys99 insome alpha interferon species such as alpha 1; SEQ ID NO: 4) is notessential for activity. The alpha 2-interferon protein does not containany N-linked glycosylation sites. The crystal structure of alphainterferon has been determined (Radhakrishnan et al., 1996).

[0125] This example provides cysteine added variants in the regionproximal to the A helix, distal to the E helix, in the A-B loop, in theB-C loop, in the C-D loop and in the D-E loop. Preferred sites for theintroduction of cysteine residues in these regions of the alphainterferon-2 species are: D2, L3, P4, Q5, T6, S8, Q20, R22, K23, S25,F27, S28, K31, D32, R33, D35, G37, F38, Q40, E41, E42, F43, G44, N45,Q46, F47, Q48, K49, A50, N65, S68, T69, K70, D71, S72, S73, A74, A75,D77, E78, T79, Y89, Q90, Q91, N93, N94, E96, A97, Q101, G102, G104,T106, E107, T108, P109, K112, E113, D114, S115, K131, K132, K133, K134,Y135, S136, A139, S152, S154, T155, N156, L157, Q158, E159, S160, L161,R162, S163, K164, E165. Variants in which cysteine residues areintroduced proximal to the first amino acid of the mature protein, i.e.,proximal to C1, or distal to the final amino acid in the mature protein,i.e., distal to E165 are provided. Other variants in which cys-1 orcys-98 (cys-99 in some alpha interferon species) have been replaced withother amino acids, preferably serine or alanine, also are provided.Other variants in which Cys-1 has been deleted (des Cys-1) also areprovided. The cysteine variants may be in the context of any naturallyoccurring or non-natural alpha interferon sequence, e.g., consensusinterferon or interferon protein hybrids. Some naturally occurring alphainterferon species (e.g., alpha interferon-1) contain a naturallyoccurring “free” cysteine. In such interferon species the naturallyoccurring free cysteine can be changed to another amino acid, preferablyserine or alanine.

[0126] This example also provides cysteine variants of other alphainterferon species, including consensus interferon, at equivalent sitesin these proteins. The alignment of the alpha interferon-2 species withother known alpha interferon species and consensus interferon is givenin Blatt et al. (1996). The crystal structure of alpha interferon-2 hasbeen determined by Rhadhakrishnan et al. (1996). Lydon et al (1985)found that deletion of the first four amino acids from the N-terminus ofalpha interferon did not affect biological activity. Valenzuela et al(1985) found that substitution of Phe-47 in alpha interferon-2 with Cys,Tyr or Ser did not alter biological activity of the protein. Cys-1 andCys-98 have been changed individually to glycine and serine,respectively, without altering biological activity of the protein(DeChiara et al, 1986).

[0127] DNA sequences encoding alpha interferon-2 can be amplified fromhuman genomic DNA, since alpha interferon genes do not contain introns(Pestka et al., 1987). The DNA sequence of alpha interferon-2 is givenin Goeddel et al. (1980). Alternatively, a cDNA for alpha interferon-2can be isolated from human lymphoblastoid cell lines that are known toexpress alpha interferon spontaneously or after exposure to viruses(Goeddell et al., 1980; Pickering et al., 1980). Many of these celllines are available from the American Type Culture Collection(Rockville, Md.). Specific mutations can be introduced into the alphainterferon sequence using plasmid-based site-directed mutagenesis kits(e.g., Quick-Change Mutagenesis Kit, Stratagene, Inc.), phagemutagenesis strategies or employing PCR mutagenesis as described for GH.

[0128] Alpha interferon has been successfully produced in E. coli as anintracellular protein (Tarnowski et al., 1986; Thatcher and Panayotatos,1986). Similar procedures can be used to express alpha interferonmuteins. Plasmids encoding alpha interferon or alpha interferon muteinscan be cloned into an E. coli expression vector such as pET15b(Novagene, Inc.) that uses the strong T7 promoter or pCYB 1 (New EnglandBioLabs, Beverly, Mass.) that uses the TAC promoter. Expression of theprotein can be induced by adding IPTG to the growth media.

[0129] Recombinant alpha interferon expressed in E. coli is sometimessoluble and sometimes insoluble (Tarnowski et al., 1986; Thatcher andPanayotatos, 1986). Insolubility appears to be related to the degree ofoverexpression of the protein. Insoluble alpha interferon proteins canbe recovered as inclusion bodies and renatured to a fully activeconformation following standard oxidative refolding protocols (Thatcherand Panayotatos, 1986; Cox et al., 1994). The alpha interteron proteinscan be purified further using other chromatographic methods such asion-exchange, hydrophobic interaction, size-exclusion and reversed phaseresins (Thatcher and Panayotaos, 1986). Protein concentrations can bedetermined using commercially available protein assay kits (Bio-RadLaboratories).

[0130] If E. coli expression of alpha interferon muteins is notsuccessful, one can express the proteins in insect cells as secretedproteins as described for GH. The proteins can be modified to containthe natural alpha interferon signal sequence (Goeddell et al., 1980) orthe honeybee mellitin signal sequence (Invitrogen, Inc.) to promotesecretion of the proteins. Alpha interferon and alpha interferon muteinscan be purified from conditioned media using conventional chromatographyprocedures. Antibodies to alpha interferon can be used in conjunctionwith Western blots to localize fractions containing the alpha interferonproteins during chromatography. Alternatively, fractions containingalpha interferon proteins can be identified using ELISAs.

[0131] Bioactivities of alpha interferon and alpha interferon muteinscan be measured using an in vitro viral plaque reduction assay (Ozes etal., 1992; Lewis, 1995). Human HeLa cells can be plated in 96-wellplates and grown to near confluency at 37° C. The cells are then washedand treated for 24 hour with different concentrations of each alphainterferon preparation. Controls should include no alpha interferon andwild type alpha interferon (commercially available from Endogen, Inc.,Woburn, Mass.). A virus such as Vesicular stomatitis virus (VSV) orencephalomyocarditis virus (EMCV) is added to the plates and the platesincubated for a further 24-48 hours at 37° C. Additional controls shouldinclude samples without virus. When 90% or more of the cells have beenkilled in the virus-treated, no alpha interferon control wells(determined by visual inspection of the wells), the cell monolayer arestained with crystal violet and absorbance of the wells read using amicroplate reader. Alternatively, the cell monolayers can be stainedwith the dye MTT (Lewis, 1995). Samples should be analyzed in duplicateor triplicate. EC₅₀ values (the amount of protein required to inhibitthe cytopathic effect of the virus by 50%) can be used to compare therelative potencies of the proteins. Wild type alpha interferon-2protects cells from the cytopathic effects of VSV and EMCV and has aspecific activity of approximately 2×10⁸ units/mg in this assay (Ozes etal., 1992). Alpha interferon muteins displaying EC₅₀ values comparableto wild type Alpha Interferon are preferable.

[0132] Alpha interferon muteins that retain activity can be PEGylatedusing procedures is similar to those described for GH. Wild type alphainterferon-2 can serve as a control since it should not PEGylate undersimilar conditions. The lowest amount of PEG that gives significantquantities of mono-pegylated product without giving di-pegylated productshould be considered optimum. Mono-PEGylated protein can be purifiedfrom non-PEGylated protein and unreacted PEG by size-exclusion or ionexchange chromatography. The purified PEGylated proteins can be testedin the viral plaque reduction bioassay described above to determinetheir bioactivities. PEGylated alpha interferon proteins withbioactivities comparable to wild type alpha interferon are preferable.Mapping the PEG attachment site and determination of pharmacokineticdata for the PEGylated protein can be performed as described for GH.

[0133] In vivo bioactivities of the PEG-alpha interferon muteins can betested using tumor xenograft models in nude mice and viral infectionmodels (Balkwill, 1986; Fish et al., 1986). Since PEG-alpha interferonbioactivity may be species-specific, one should confirm activity of thePEGylated protein using appropriate animal cell lines in in vitro virusplaque reduction assays, similar to those described above. Next, oneshould explore the effects of different dosing regimens and differenttimes of injections to determine if PEG-alpha interferon is more potentand produces longer lasting effects than non-PEGylated alpha interferon.

[0134] The novel alpha interferon-derived molecules of this example canbe formulated and tested for activity essentially as set forth inExamples 1 and 2, substituting, however, the appropriate assays andother considerations known in the art related to the specific proteinsof this example.

EXAMPLE 4 Beta Interferon

[0135] Beta interferon is produced by fibroblasts and exhibitsantiviral, antitumor and immunomodulatory effects. The single-copy betainterferon gene encodes a preprotein that is cleaved to yield a matureprotein of 166 amino acids (Taniguchi et al. 1980; SEQ ID NO: 5). Theprotein contains three cysteines, one of which, cysteine-17, is “free”,i.e., it does not participate in a disulfide bond. The protein containsone N-linked glycosylation site. The crystal structure of the proteinhas been determined (Karpusas et al. 1997)

[0136] This example provides cysteine-added variants at any of the threeamino acids that comprise the N-linked glycosylation sites, i.e., N80C,E81C or T82C. This example also provides cysteine-added variants in theregion proximal to the A helix, distal to the E helix, in the A-B loop,in the B-C loop, in the C-D loop and in the D-E loop. Preferred sitesfor introduction of cysteine residues in these regions are: M1, S2, Y3,N4, L5, Q23, N25, G26, R27, E29, Y30, K33, D34, R35, N37, D39, E42, E43,K45, Q46, L47, Q48, Q49, Q51, K52, E53, A68, F70, R71, Q72, D73, S74,S75, S76, T77, G78, E107, K108, E109, D110, F111, T112, R113, G114,K115, L116, A135, K136, E137, K138, S139, I157, N158, R159, L160, T161,G162, Y163, L164, R165 and N166. Variants in which cysteine residues areintroduced proximal to the first amino acid of the mature protein, i.e.,proximal to M1, or distal to the final amino acid in the mature protein,i.e., distal to N166 also are provided.

[0137] These variants are produced in the context of the natural proteinsequence or a variant protein in which the naturally occurring “free”cysteine residue (cysteine-17) has been changed to another amino acid,preferably serine or alanine.

[0138] The novel beta interferon-derived molecules of this example canbe formulated and tested for activity essentially as set forth inExamples 1 and 2, substituting, however, the appropriate assays andother considerations known in the art related to the specific proteinsof this example.

EXAMPLE 5 Granulocyte Colony-Stimulating Factor (G-CSF)

[0139] G-CSF is a pleuripotent cytokine that stimulates theproliferation, differentiation and function of granulocytes. The proteinis produced by activated monocytes and macrophages. The amino acidsequence of G-CSF (SEQ ID NO: 6) is given in Souza et al. (1986), Nagataet al. (1986a, b) and U.S. Pat. No. 4,810,643 all incorporated herein byreference. The human protein is synthesized as a preprotein of 204 or207 amino acids that is cleaved to yield mature proteins of 174 or 177amino acids. The larger form has lower specific activity than thesmaller form. The protein contains five cysteines, four of which areinvolved in disulfide bonds. Cysteine-17 is not involved in a disulfidebond. Substitution of cysteine-17 with serine yields a mutant G-CSFprotein that is fully active (U.S. Pat. No. 4,810,643). The protein isO-glycosylated at threonine-133 of the mature protein.

[0140] Hill et al., Proceedings of the National Academy of Science vol.90: 5167-5171 (1993) identifies the amino acids that comprise HelicesA-D of G-CSF (see the legend to FIG. 2of this publication). Thepositions of the four helical regions and the intervening (loop)regions, as well as the regions preceding Helix A and following Helix D,are located within the sequence for human granulocyte colony-stimulatingfactor at the following positions (positions given relative to SEQ IDNO:6):

[0141] Preceding Helix A=residues 1-10;

[0142] Helix A=residues 11-39;

[0143] A-B loop=residues 40-70;

[0144] Helix B=residues 71-91;

[0145] B-C loop=residues 92-99;

[0146] Helix C=residues 100-123;

[0147] C-D loop=residues 124-142;

[0148] Helix D=residues 143-17;

[0149] Following Helix D=residues 173-174.

[0150] This example provides a cysteine-added variant at threonine-133.This example provides other cysteine-added variants in the regionproximal to helix A, distal to helix D, in the A-B loop, B-C loop andC-D loop. Preferred sites for introduction of cysteine substitutions inthese regions are: T1, P2, L3, G4, P5, A6, S7, S8, L9, P10, Q11, S12,T38, K40, S53, G55, W58, A59, P60, S62, S63, P65, S66, Q67, A68, Q70,A91, E93, G94, S96, E98, G100, G125, M126, A127, A129, Q131, T133, Q134,G135, A136, A139, A141, S142, A143, Q145, Q173 and P174. Variants inwhich cysteine residues are introduced proximal to the first amino acidof the mature protein, i.e., proximal to T1, or distal to the finalamino acid in the mature protein, i.e., distal to P174 are provided.These variants are provided in the context of the natural proteinsequence or a variant protein in which the naturally occurring “free”cysteine residue (cysteine-17) has been changed to another amino acid,preferably serine or alanine.

[0151] A cDNA encoding human G-CSF can be purchased from R&D Systems(Minneapolis, Minn.) or amplified using PCR from mRNA isolated fromhuman carcinoma cell lines such as 5637 and U87-MG known to expressG-CSF constitutively (Park et al., 1989; Nagata, 1994). These cell linesare available from the American Type Culture collection (Rockville,Md.). Specific mutations can be introduced into the G-CSF sequence usingplasmid-based site-directed mutagenesis kits (e.g., Quick-ChangeMutagenesis Kit, Stratagene, Inc.), phage mutagenesis methods oremploying PCR mutagenesis as described for GH.

[0152] G-CSF has been successfully produced in E. coli as anintracellular protein (Souza et al., 1986). One can employ similarprocedures to express G-CSF and G-CSF muteins. Plasmids encoding G-CSFor G-CSF muteins can be cloned into an E. coli expression vector such aspET15b (available from Novagen, Inc., Madison, Wis.) that uses thestrong T7 promoter or pCYB1 (available from New England BioLabs,Beverly, Mass.) that uses the strong TAC promoter. Expression of theprotein can be induced by adding IPTG to the growth media. RecombinantG-CSF expressed in E. coli is insoluble and can be recovered asinclusion bodies. The protein can be renatured to a fully activeconformation following standard oxidative refolding protocols (Souza etal., 1986; Lu et al., 1992; Cox et al., 1994). Similar procedures can beused to refold cysteine muteins. The proteins can be purified furtherusing other chromatographic methods such as ion exchange, hydrophobicinteraction, size-exclusion and reversed phase resins (Souza et al.,1986; Kuga et al., 1989; Lu et al., 1992). Protein concentrations can bedetermined using commercially available protein assay kits (Bio-RadLaboratories).

[0153] If E. coli expression of G-CSF or G-CSF muteins is notsuccessful, one can express G-CSF and G-CSF muteins in insect cells assecreted proteins as described for GH. The proteins can be modified tocontain the natural G-CSF signal sequence (Souza et al., 1986; Nagata etal., 1986a; Nagata et al, 1986b) or the honeybee mellitin signalsequence (Invitrogen, Inc., Carlsbad, Calif.) to promote secretion ofthe proteins. G-CSF and G-CSF muteins can be purified from conditionedmedia using conventional chromatography procedures. Antibodies to G-CSFcan be used in conjunction with Western blots to localize fractionscontaining the G-CSF proteins during chromatography. Alternatively,fractions containing G-CSF proteins can be identified using ELISAs.

[0154] G-CSF muteins also can be expressed in mammalian cells asdescribed for erythropoietin in Example 2.

[0155] Bioactivities of G-CSF and the G-CSF muteins can be measuredusing an in vitro cell proliferation assay. The mouse NFS-60 cell lineand the human AML-193 cell line can be used to measure G-CSF bioactivity(Tsuchiya et al., 1986; Lange et al., 1987; Shirafuji et al., 1989).Both cell lines proliferate in response to human G-CSF. The AML-193 cellline is preferable since it is of human origin, which eliminates thepossibility of a false conclusion resulting from species differences.The NFS60 cell lines proliferates in response to G-CSF with ahalf-maximal effective concentration (EC₅₀) of 10-20 picomolar. PurifiedG-CSF and G-CSF muteins can be tested in cell proliferation assays usingthese cell lines to determinc specific activities of the proteins, usingpublished methods (Tsuchiya et al., 1986; Lange et al., 1987; Shirafujiet al., 1989). Cells can be plated in 96-well tissue culture dishes withdifferent concentrations of G-CSF or G-CSF muteins. After 1-3 days at37° C. in a humidified tissue culture incubator, proliferation can bemeasured by ³H-thymidine incorporation as described for GH. Assaysshould be performed at least three times for each mutein usingtriplicate wells for each data point. EC₅₀ values can be used to comparethe relative potencies of the muteins. G-CSF muteins displaying similaroptimal levels of stimulation and EC₅₀ values comparable to wild typeG-CSF are preferable.

[0156] G-CSF muteins that retain activity can be PEGylated usingprocedures similar to those described for GH. Wild type G-CSF and ser-17G-CSF can serve as controls since they should not PEGylate under similarconditions. The lowest amount of PEG that gives significant quantitiesof mono-PEGylated product without giving di-PEGylated product should beconsidered optimum. Mono-PEGylated protein can be purified fromnon-PEGylated protein and unreacted PEG by size-exclusion or ionexchange chromatography. The purified PEGylated proteins can be testedin the cell proliferation assay described above to determine theirbioactivities.

[0157] The PEG site in the protein can be mapped using proceduressimilar to those described for GH. Pharmacokinetic data for thePEGylated proteins can be obtained using procedures similar to thosedescribed for GH.

[0158] Initial studies to demonstrate in vivo efficacy of PEG-G-CSF canbe done in normal Sprague-Dawley rats, which can be purchased fromCharles River. Groups of rats should receive single subcutaneous orintravenous injections of various doses of G-CSF, PEG-G-CSF or placebo.Animals should be sacrificed at daily intervals for up to a week fordetermination of neutrophil and total white blood cell counts inperipheral blood. Other blood cell types (platelets and red blood cells)can be measured to demonstrate cell specificity.

[0159] The efficacy of PEG-G-CSF can be tested in a rat neutropeniamodel. Neutropenia can be induced by treatment with cyclophosphamide,which is a commonly used chemotherapeutic agent that ismyelosuppressive. G-CSF accelerates recovery of normal neutrophil levelsin cyclophosphamide-treated animals (Kubota et al., 1990). Rats receivean injection of cyclophosphamide on day 0 to induce neutropenia. Theanimals are then be divided into different groups, which will receivesubcutaneous injections of G-CSF, PEG-G-CSF or placebo. Neutrophil andtotal white blood cell counts in peripheral blood should be measureddaily until they return to normal levels. Initially one should confirmthat G-CSF accelerates recovery from neutropenia when injected daily.Next, one should explore the effects of different dosing regimens anddifferent times of injections to determine if PEG-G-CSF is more potentand produces longer lasting effects than non-PEGylated G-CSF.

[0160] The novel GCSF-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 6 Thrombopoietin (TPO)

[0161] Thrombopoietin stimulates the development of megakaryocyteprecursors of platelets. The amino acid sequence of TPO (SEQ ID NO: 7)is given in Bartley et al.(1994), Foster et al.(l 994), de Sauvage etal.(1994), each incorporated herein by reference.

[0162] The protein is synthesized as a 353 amino acid precursor proteinthat is cleaved to yield a mature protein of 332 amino acids. TheN-terminal 154 amino acids have homology with EPO and other members ofthe GH supergene family. The C-terminal 199 amino acids do not sharehomology with any other known proteins. The C-terminal region containssix N-linked glycosylation sites and multiple O-linked glycosylationsites (Hoffman et al., 1996). O-linked glycosylation sites also arefound in the region proximal to the A helix, in the A-B loop, and at theC-terminus of Helix C (Hoffman et al., 1996). A truncated TPO proteincontaining only residues 1-195 of the mature protein is fully active invitro (Bartley et al., 1994).

[0163] This example provides cysteine-added variants at any of the aminoacids that comprise the N-linked glycosylation sites and the O-linkedglycosylation sites. This example also provides cysteine-added variantsin the region proximal to the A helix, distal to the D helix, in the A-Bloop, in the B-C loop, and in the C-D loop.

[0164] Preferred sites for introduction of cysteine residues are: S1,P2, A3, P4, P5, A6, T37, A43, D45, S47, G49, E50, K52, T53, Q54, E56,E57, T58, A76, A77, R78, G79, Q80, G82, T84, S87, S88, G109, T110, Q111,P113, P114, Q115, G116, R117, T118, T119, A120, H121, K122, G146, G147,S148, T149, A155, T158,T159, A160, S163, T165, S166, T170, N176, R177,T178, S179, G180, E183, T184, N185, F186, T187, A188, S189, A190, T192,T193, G194, S195, N213, Q214, T215, S216, S218, N234, G235, T236, S244,T247, S254, S255, T257, S258, T260, S262, S272, S274, T276, T280, T291,T294, S307, T310, T312, T314, S315, N319, T320, S321, T323, S325, Q326,N327, L328, S329, Q330, E331, and G332. Variants in which cysteineresidues are introduced proximal to the first amino acid of the matureprotein, i.e., proximal to S1, or distal to the final amino acid in themature protein, i.e., distal to G332 are provided. The cysteine-addedvariants are provided in the context of the natural human protein or avariant protein that is truncated between amino acids 147 and theC-terminus of the natural protein, G332. Variants in which cysteineresidues are added distal to the final amino acid of a TPO protein thatis truncated between amino acids 147 and 332 also are provided.

[0165] The novel TPO-derived molecules of this example can be formulatedand tested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 7 Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF)

[0166] GM-CSF stimulates the proliferation and differentiation ofvarious hematopoietic cells, including neutrophil, monocyte, eosinophil,erythroid, and megakaryocyte cell lineages. The amino acid sequence ofhuman GM-CSF (SEQ ID NO: 8) is given in Cantrell et al. (1985) and Leeet al (1985) both incorporated herein by reference.

[0167] GM-CSF is produced as a 144 amino acid preprotein that is cleavedto yield a mature 127 amino acid protein. The mature protein has twosites for N-linked glycosylation. One site is located at the C-terminalend of Helix A; the second site is in the A-B loop.

[0168] This example provides cysteine-added variants at any of the aminoacids that comprise the N-linked glycosylation sites, i.e., N27C, L28C,S29C, N37C, E38C and T39C. This example also provides cysteine-addedvariants in the region proximal to the A helix, distal to the D helix,in the A-B loop, in the B-C loop, and in the C-D loop. Preferred sitesfor introduction of cysteine substitutions in these regions are: A1, P2,A3, R4, S5, P6, S7, P8, S9, T10, Q11, R30, D31, T32, A33, A34, E35, E41,S44, E45, D48, Q50, E51, T53, Q64, G65, R67, G68, S69, L70, T71, K72,K74, G75, T91, E93, T94, S95, A97, T98, T102, I117, D120, E123, V125,Q126 and E127. Variants in which cysteine residues are introducedproximal to the first amino acid of the mature protein, i.e., proximalto A1, or distal to the final amino acid in the mature protein, i.e.,distal to E127 are provided.

[0169] The novel GM-CSF-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 8 IL-2

[0170] IL-2 is a T cell growth factor that is synthesized by activated Tcells. The protein stimulates clonal expansion of activated T cells.Human IL-2 is synthesized as a 153 amino acid precursor that is cleavedto yield a 133 amino acid mature protein (Tadatsugu et al., 1983; Devoset al., 1983; SEQ ID NO: 9).

[0171] The amino acid sequence of IL-2 is set forth in (Tadatsugu et al.1983; Devos et al. 1983). The mature protein contains three cysteineresidues, two of which form a disulfide bond. Cysteine-125 of the matureprotein is not involved in a disulfide bond. Replacement of cysteine-125with serine yields an IL-2 mutein with full biological activity (Wang etal., 1984). The protein is O-glycosylated at threonine-3 of the matureprotein chain.

[0172] This example provides cysteine-added variants in the last fourpositions of the D helix, in the region distal to the D helix, in theA-B loop, in the B-C loop, and in the C-D loop. These variants areprovided in the context of the natural protein sequence or a variantprotein in which the naturally occurring “free” cysteine residue(cysteine-125) has been changed to another amino acid, preferably serineor alanine. Variants in which cysteine residues are introduced proximalto the first amino acid, i.e., A1, or distal to the final amino acid,i.e., T133, of the mature protein, also are provided.

[0173] The novel IL-2-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 9 IL-3

[0174] IL-3 is produced by activated T cells and stimulates theproliferation and differentiation of pleuripotent hematopoietic stemcells. The amino acid sequence of human IL-3 (SEQ ID NO: 10) is given inYang et al. (1986); Dorssers et al. (1987) and Otsuka et al. (1988) allincorporated herein by reference. The protein contains two cysteineresidues and two N-linked glycosylation sites. Two alleles have beendescribed, resulting in isoforms having serine or proline at amino acidposition 8 or the mature protein.

[0175] This example provides cysteine-added variants at any of the aminoacids that comprise the N-linked glycosylation sites. This example alsoprovides cysteine-added variants in the region proximal to the A helix,distal to the D helix, in the A-B loop, in the B-C loop, and in the C-Dloop. Variants in which cysteine residues are introduced proximal to thefirst amino acid or distal to the final amino acid in the matureprotein, also are provided.

[0176] The novel IL-3-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 10 IL-4

[0177]

[0178] IL-4 is a pleiotropic cytokine that stimulates the proliferationand differentiation of monocytes and T and B cells. IL-4 has beenimplicated in the process that leads to B cells secreting IgE, which isbelieved to play a role in asthma and atopy. The bioactivity of IL-4 isspecies specific. IL-4 is synthesized as a 153 amino acid precursorprotein that is cleaved to yield a mature protein of 129 amino acids.The amino acid sequence of human IL-4 (SEQ ID NO: 11) is given in Yokotaet al. (1986) which is incorporated herein by reference. The proteincontains six cysteine residues and two N-linked glycosylation sites. Theglycosylation sites are located in the A-B and C-D loops.

[0179] This example provides cysteine-added variants at any of the aminoacids that comprise the N-linked glycosylation sites. This, example alsoprovides cysteine-added variants in the region proximal to the A helix,distal to the D helix, in the A-B loop, in the B-C loop, and in the C-Dloop. Variants in which cysteine residues are introduced proximal to thefirst amino acid, H1, or distal to the final amino acid, S129, of themature protein are provided.

[0180] The novel IL-4-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 11 IL-5

[0181] IL-5 is a differentiation and activation factor for eosinophils.The amino acid sequence of human IL-5 (SEQ ID NO: 12) is given in Yokotaet al. (1987) which is incorporated herein by reference. The matureprotein contains 115 amino acids and exists in solution as adisulfide-linked homodimer. The protein contains both O-linked andN-linked glycosylation sites.

[0182] This example provides cysteine-added variants in the regionproximal to the A helix, distal to the D helix, in the A-B loop, in theB-C loop, and in the C-D loop. Variants in which cysteine residues areadded proximal to the first amino acid or distal to the final amino acidin the mature protein are provided.

[0183] The novel IL-5-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 12 IL-6

[0184] IL-6 stimulates the proliferation and differentiation of manycell types. The amino acid sequence of human IL-6 (SEQ ID NO: 13) isgiven in Hirano et al. (1986) which is incorporated herein by reference.IL-6 is synthesized as a 212 amino acid preprotein that is cleaved togenerate a 184 amino acid mature protein. The mature protein containstwo sites for N-linked glycosylation and one site for O-glycosylation,at T137, T138, T142 or T143.

[0185] This example provides cysteine-added variants at any of the aminoacids that comprise the N-linked glycosylation sites and at the O-linkedglycosylation site. Also provided are cysteine-added variants in theregion proximal to the A helix, distal to the D nelix, in the A-B loop,in the B-C loop, and in the C-D loop. Variants in which cysteineresidues are added proximal to the first amino acid or distal to thefinal amino acid of the mature protein also are provided.

[0186] The novel IL-6-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 13 IL-7

[0187] IL-7 stimulates proliferation of immature B cells and acts onmature T cells. The amino acid sequence of human IL-7 (SEQ ID NO: 14) isgiven in Goodwin et al. (1989) which is incorporated herein byreference. The protein is synthesized as a 177 amino acid preproteinthat is cleaved to yield a 152 amino acid mature protein that containsthree sites for N-linked glycosylation.

[0188] This example provides cysteine-added variants at any of the aminoacids that comprise the N-linked glycosylation sites. This example alsoprovides cysteine-added variants in the region proximal to the A helix,distal to the D helix, in the A-B loop, in the B-C loop, and in the C-Dloop.

[0189] The novel IL-7-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample. Variants in which cysteine residues are added proximal to thefirst amino acid or distal to the final amino acid of the mature proteinalso are provided.

EXAMPLE 14 IL-9

[0190] IL-9 is a pleiotropic cytokine that acts on many cell types inthe lymphoid, myeloid and mast cell lineages. IL-9 stimulates theproliferation of activated T cells and cytotoxic T lymphocytes,stimulates proliferation of mast cell precursors and synergizes witherythropoietin in stimulating immature red blood cell precursors. Theamino acid sequence of human IL-9 (SEQ ID NO: 15) is given in Yang etal. (1989) which is incorporated herein by reference. IL-9 issynthesized as a precursor protein of 144 amino acids that is cleaved toyield a mature protein of 126 amino acids. The protein contains fourpotential N-linked glycosylation sites.

[0191] This example provides cysteine-added variants at any of the threeamino acids that comprise the N-linked glycosylation sites. This examplealso provides cysteine-added variants in the region proximal to the Ahelix, distal to the D helix, in the A-B loop, in the B-C loop, and inthe C-D loop. Variants in which cysteine residues are added proximal tothe first amino acid or distal to the final amino acid of the matureprotein also are provided.

[0192] The novel IL-9-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 15 IL-10

[0193] The amino acid sequence of human IL-10 (SEQ ID NO: 16) is givenin Vieira et al. (1991) which is incorporated herein by reference. IL-10is synthesized as a 178 amino acid precursor protein that is cleaved toyield a mature protein of 160 amino acids. IL-10 can function toactivate or suppress the immune system. The protein shares structuralhomology with the interferons, i.e., it contains five amphipathichelices. The protein contains one N-linked glycosylation site.

[0194] This example provides cysteine-added variants at any of the threeamino acids comprising the N-linked glycosylation site. This examplealso provides cysteine-added variants in the region proximal to the Ahelix, distal to the E helix, in the A-B loop, in the B-C loop, in theC-D loop and in the D-E loop. Variants in which cysteine residues areadded proximal to the first amino acid or distal to the final amino acidof the mature protein also are provided.

[0195] The novel IL-10-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 16 IL-11

[0196] IL-11 is a pleiotropic cytokine that stimulates hematopoiesis,lymphopoeisis and acute phase responses. IL-11 shares many biologicaleffects with IL-6. The amino acid sequence of human IL-11 (SEQ ID NO:17) is given in Kawashima et al. (1991) and Paul et al. (1990) bothincorporated herein by reference. IL-11 is synthesized as a precursorprotein of 199 amino acids that is cleaved to yield a mature protein of178 amino acids. There are no N-linked glycosylation sites in theprotein.

[0197] This example provides cysteine-added variants in the regionproximal to the A helix, distal to the D helix, in the A-B loop, in theB-C loop, and in the C-D loop. Variants in which cysteine residues areadded proximal to the first amino acid or distal to the final amino acidof the mature protein also are provided.

[0198] The novel IL-11-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 17 IL-12 p35

[0199] IL-12 stimulates proliferation and differentiation of NK cellsand cytotoxic T lymphocytes. IL-12 exists as a heterodimer of a p35subunit and a p40 subunit. The p35 subunit is a member of the GHsupergene family. The amino acid sequence of the p35 subunit (SEQ ID NO:18) is given in Gubler et al. (1991) and Wolf et al. (1991) bothincorporated herein by reference. p35 is synthesized as a precursorprotein of 197 amino acids and is cleaved to yield a mature protein of175 amino acids. The protein contains 7 cysteine residues and threepotential N-linked glycosylation sites.

[0200] This example provides cysteine-added variants at any of the threeamino acids that comprise the three N-linked glycosylation sites. Thisexample also provides cysteine-added variants in the region proximal tothe A helix, distal to the D helix, in the A-B loop, in the B-C loop,and in the C-D loop. These variants are provided in the context of thenatural protein sequence or a variant protein in which the naturallyoccurring “free” cysteine residue has been changed to another aminoacid, preferably serine or alanine. Variants in which cysteine residuesare added proximal to the first amino acid or distal to the final aminoacid of the mature protein also are provided.

[0201] The novel IL-12 p35-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 18 IL-13

[0202] IL-13 shares many biological properties with IL-4. The amino acidsequence of human IL-13 (SEQ ID NO: 19) is given in McKenzie et al.(1993) and Minty et al. (1993) both incorporated herein by reference.The protein is synthesized as a 132 amino acid precursor protein that iscleaved to yield a mature protein of 112 amino acids. The mature proteincontains 5 cysteine residues and multiple N-linked glycosylation sites.A variant in which glutamine at position 78 is deleted due toalternative mRNA splicing has been described (McKenzie et al 1993)

[0203] This example provides cysteine-added variants at any of the threeamino acids comprising the N-linked glycosylation sites. This examplealso provides cysteine-added variants in the region proximal to the Ahelix, distal to the D helix, in the A-B loop, in the B-C loop, and inthe C-D loop. These variants are provided in the context of the naturalprotein sequence or a variant sequence in which the pre-existing “free”cysteine has been changed to another amino acid, preferably to alanineor serine. Variants in which cysteine residues are added proximal to thefirst amino acid or distal to the final amino acid of the mature proteinalso are provided.

[0204] The novel IL-13-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 19 IL-15

[0205] IL-15 stimulates the proliferation and differentiation of Tcells, NK cells, LAK cells and Tumor Infiltrating Lymphocytes. IL-15 maybe useful for treating cancer and viral infections. The sequence ofIL-15 (SEQ ID NO: 20) is given in Anderson et al. (1995) which isincorporated herein by reference. IL-15 contains two N-linkedglycosylation sites, which are located in the C-D loop and C-terminalend of the D helix. IL-15 encodes a 162 amino acid preprotein that iscleaved to generate a mature 114 amino acid protein.

[0206] This example provides cysteine-added variants at any of the threeamino acids comprising the N-linked glycosylation sites in the C-D loopor C-terminal end of the D helix. This example also providescysteine-added variants proximal to helix A, in the A-B loop, the B-Cloop, the C-D loop or distal to helix D. Variants in which cysteineresidues are added proximal to the first amino acid or distal to thefinal amino acid of the mature protein also are provided.

[0207] The novel IL-15-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 20 Macrophage Colony Stimulating Factor (M-CSF)

[0208] M-CSF regulates the growth, differentiation and function ofmonocytes. The protein is a disulfide-linked homodimer. Multiplemolecular weight species of M-CSF, which arise from differential mRNAsplicing, have been described. The amino acid sequence of human M-CSFand its various processed forms are given in Kawasaki et al (1985), Wonget al. (1987) and Cerretti et al (1988) which are incorporated herein byreference. Cysteine-added variants can be produced following the generalteachings of this application and in accordance with the examples setforth herein.

[0209] The novel MCSF-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 21 Oncostatin M

[0210] Oncostatin M is a multifunctional cytokine that affects thegrowth and differentiation of many cell types. The amino acid sequenceof human oncostatin M (SEQ ID NO: 21) is given in Malik et al. (1989)which is incorporated herein by reference. Oncostatin M is produced byactivated monocytes and T lymphocytes. Oncostatin M is synthesized as a252 amino acid preprotein that is cleaved sequentially to yield a 227amino acid protein and then a 196 amino acid protein (Linsley et al.,1990). The mature protein contains O-linked glycosylation sites and twoN-linked glycosylation sites. The protein is O-glycosylated at T160,T162 and S165. The mature protein contains five cysteine residues.

[0211] This example provides cysteine-added variants at either of thethree amino acids comprising the N-linked glycosylation sites or at theamino acids that comprise the O-linked glycosylation sites. This examplealso provides cysteine-added variants proximal to helix A, in the A-Bloop, in the B-C loop, in the C-D loop or distal to helix D. Thesevariants are provided in the context of the natural protein sequence ora variant sequence in which the pre-existing “free” cysteine has beenchanged to another amino acid, preferably to alanine or serine. Variantsin which cysteine residues are added proximal to the first amino acid ordistal to the final amino acid of the mature protein also are provided.

[0212] The novel Oncostatin M-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 22 Ciliary Neurotrophic Factor (CNTF)

[0213] The amino acid sequence of human CNTF (SEQ ID NO: 22) is given inLam et al. (1991) which is incorporated herein by reference. CNTF is a200 amino acid protein that contains no glycosylation sites or signalsequence for secretion. The protein contains one cysteine residue. CNTFfunctions as a survival factor for nerve cells.

[0214] This example provides cysteine-added variants proximal to helixA, in the A-B loop, the B-C loop, the C-D loop or distal to helix D.These variants are provided in the context of the natural proteinsequence or a variant sequence in which the pre-existing “free” cysteinehas been changed to another amino acid, preferably to alanine or serine.Variants in which cysteine residues are added proximal to the firstamino acid or distal to the final amino acid of the mature protein alsoare provided.

[0215] The novel CNTF-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 23 Leukemia Inhibitory Factor (LIF)

[0216] The amino acid sequence of LIF (SEQ ID NO: 23) is given in Moreauet al. (1988) and Gough et al. (1988) both incorporated herein byreference. The human gene encodes a 202 amino acid precursor that iscleaved to yield a mature protein of 180 amino acids. The proteincontains six cysteine residues, all of which participate in disulfidebonds. The protein contains multiple O— and N-linked glycosylationsites. The crystal structure of the protein was determined by Robinsonet al. (1994). The protein affects the growth and differentiation ofmany cell types.

[0217] This example provides cysteine-added variants at any of the threeamino acids comprising the N-linked glycosylation sites or the O-linkedglycosylation site. Also provided are cysteine-added variants proximalto helix A, in the A-B loop, the B-C loop, the C-D loop or distal tohelix D. Variants in which cysteine residues are added proximal to thefirst amino acid or distal to the final amino acid of the mature proteinalso are provided.

[0218] The novel LIF-derived molecules of this example can be formulatedand tested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

[0219] All of the documents cited herein are incorporated herein byreference.

[0220] The protein analogues disclosed herein can be used for the knowntherapeutic uses of the native proteins in essentially the same formsand doses all well known in the art.

[0221] While the exemplary preferred embodiments of the presentinvention are described herein with particularity, those having ordinaryskill in the art will recognize changes, modifications, additions, andapplications other than those specifically described herein, and mayadapt the preferred embodiments and methods without departing from thespirit of this invention.

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1 41 1 191 PRT Homo sapiens 1 Phe Pro Thr Ile Pro Leu Ser Arg Leu PheAsp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala PheAsp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln LysTyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu SerIle Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn LeuGlu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu GluPro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr GlyAla Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu GluGly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro ArgThr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr AsnSer His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu LeuTyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu ArgIle Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 180 185 190 2 166PRT Homo sapiens 2 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu GluArg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr GlyCys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp ThrLys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln AlaVal Glu Val Trp 50 55 60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu ArgGly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser Ser Gln Pro Trp Glu Pro LeuGln Leu His Val Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr ThrLeu Leu Arg Ala Leu 100 105 110 Gly Ala Gln Lys Glu Ala Ile Ser Pro ProAsp Ala Ala Ser Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp ThrPhe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg Gly LysLeu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly Asp Arg165 3 165 PRT Homo sapiens 3 Cys Asp Leu Pro Gln Thr His Ser Leu Gly SerArg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser LeuPhe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu GluPhe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His GluMet Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser AlaAla Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu TyrGln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly ValThr Glu Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val ArgLys Tyr Phe Gln Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys TyrSer Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg SerPhe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser LysGlu 165 4 166 PRT Homo sapiens 4 Cys Asp Leu Pro Glu Thr His Ser Leu AspAsn Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Ser Arg Ile SerPro Ser Ser Cys Leu Met Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln GluGlu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys Ala Pro Ala Ile Ser Val LeuHis Glu Leu Ile Gln Gln Ile 50 55 60 Phe Asn Leu Phe Thr Thr Lys Asp SerSer Ala Ala Trp Asp Glu Asp 65 70 75 80 Leu Leu Asp Lys Phe Cys Thr GluLeu Tyr Gln Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu GluArg Val Gly Glu Thr Pro Leu Met 100 105 110 Asn Ala Asp Ser Ile Leu AlaVal Lys Lys Tyr Phe Arg Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu LysLys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile MetArg Ser Leu Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 Arg Leu ArgArg Lys Glu 165 5 166 PRT Homo sapiens 5 Met Ser Tyr Asn Leu Leu Gly PheLeu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Cys Gln Lys Leu Leu Trp GlnLeu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe AspIle Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp AlaAla Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe ArgGln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Val Glu AsnLeu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val LeuGlu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu MetSer Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His TyrLeu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val ArgVal Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 ThrGly Tyr Leu Arg Asn 165 6 174 PRT Homo sapiens 6 Thr Pro Leu Gly Pro AlaSer Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 Cys Leu Glu Gln ValArg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 Glu Lys Leu Cys AlaThr Tyr Lys Leu Cys His Pro Glu Glu Leu Val 35 40 45 Leu Leu Gly His SerLeu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys 50 55 60 Pro Ser Gln Ala LeuGln Leu Ala Gly Cys Leu Ser Gln Leu His Ser 65 70 75 80 Gly Leu Phe LeuTyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser 85 90 95 Pro Glu Leu GlyPro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp 100 105 110 Phe Ala ThrThr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro 115 120 125 Ala LeuGln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 130 135 140 GlnArg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe 145 150 155160 Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 165 170 7332 PRT Homo sapiens 7 Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val LeuSer Lys Leu Leu 1 5 10 15 Arg Asp Ser His Val Leu His Ser Arg Leu SerGln Cys Pro Glu Val 20 25 30 His Pro Leu Pro Thr Pro Val Leu Leu Pro AlaVal Asp Phe Ser Leu 35 40 45 Gly Glu Trp Lys Thr Gln Met Glu Glu Thr LysAla Gln Asp Ile Leu 50 55 60 Gly Ala Val Thr Leu Leu Leu Glu Gly Val MetAla Ala Arg Gly Gln 65 70 75 80 Leu Gly Pro Thr Cys Leu Ser Ser Leu LeuGly Gln Leu Ser Gly Gln 85 90 95 Val Arg Leu Leu Leu Gly Ala Leu Gln SerLeu Leu Gly Thr Gln Leu 100 105 110 Pro Pro Gln Gly Arg Thr Thr Ala HisLys Asp Pro Asn Ala Ile Phe 115 120 125 Leu Ser Phe Gln His Leu Leu ArgGly Lys Val Arg Phe Leu Met Leu 130 135 140 Val Gly Gly Ser Thr Leu CysVal Arg Arg Ala Pro Pro Thr Thr Ala 145 150 155 160 Val Pro Ser Arg ThrSer Leu Val Leu Thr Leu Asn Glu Leu Pro Asn 165 170 175 Arg Thr Ser GlyLeu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr 180 185 190 Thr Gly SerGly Leu Leu Lys Trp Gln Gln Gly Phe Arg Ala Lys Ile 195 200 205 Pro GlyLeu Leu Asn Gln Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly 210 215 220 TyrLeu Asn Arg Ile His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe 225 230 235240 Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser Gly 245250 255 Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly Tyr Ser260 265 270 Pro Ser Pro Thr His Pro Pro Thr Gly Gly Tyr Thr Leu Phe ProLeu 275 280 285 Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu His Pro LeuLeu Pro 290 295 300 Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser Pro LeuLeu Asn Thr 305 310 315 320 Ser Tyr Thr His Ser Gln Asn Leu Ser Gln GluGly 325 330 8 127 PRT Homo sapiens 8 Ala Pro Ala Arg Ser Pro Ser Pro SerThr Gln Pro Trp Glu His Val 1 5 10 15 Asn Ala Ile Gln Glu Ala Arg ArgLeu Leu Asn Leu Ser Arg Asp Thr 20 25 30 Ala Ala Glu Met Asn Glu Thr ValGlu Val Ile Ser Glu Met Phe Asp 35 40 45 Leu Gln Glu Pro Thr Cys Leu GlnThr Arg Leu Glu Leu Tyr Lys Gln 50 55 60 Gly Leu Arg Gly Ser Leu Thr LysLeu Lys Gly Pro Leu Thr Met Met 65 70 75 80 Ala Ser His Tyr Lys Gln HisCys Pro Pro Thr Pro Glu Thr Ser Cys 85 90 95 Ala Thr Gln Ile Ile Thr PheGlu Ser Phe Lys Glu Asn Leu Lys Asp 100 105 110 Phe Leu Leu Val Ile ProPhe Asp Cys Trp Glu Pro Val Gln Glu 115 120 125 9 133 PRT Homo sapiens 9Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 1015 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 2530 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 4045 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 5560 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 7075 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 8590 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln SerIle 115 120 125 Ile Ser Thr Leu Thr 130 10 152 PRT Homo sapiens 10 MetSer Arg Leu Pro Val Leu Leu Leu Leu Gln Leu Leu Val Arg Pro 1 5 10 15Gly Leu Gln Ala Pro Met Thr Gln Thr Thr Pro Leu Lys Thr Ser Trp 20 25 30Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln 35 40 45Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln 50 55 60Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe 65 70 7580 Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile 85 9095 Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr 100105 110 Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg115 120 125 Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala GlnGln 130 135 140 Thr Thr Leu Ser Leu Ala Ile Phe 145 150 11 129 PRT Homosapiens 11 His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu AsnSer 1 5 10 15 Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val ThrAsp Ile 20 25 30 Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe CysArg Ala 35 40 45 Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu Lys AspThr Arg 50 55 60 Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg His Lys GlnLeu Ile 65 70 75 80 Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly LeuAla Gly Leu 85 90 95 Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr LeuGlu Asn Phe 100 105 110 Leu Glu Arg Leu Lys Thr Ile Met Arg Glu Lys TyrSer Lys Cys Ser 115 120 125 Ser 12 134 PRT Homo sapiens 12 Met Arg MetLeu Leu His Leu Ser Leu Leu Ala Leu Gly Ala Ala Tyr 1 5 10 15 Val TyrAla Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val Lys Glu 20 25 30 Thr LeuAla Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala Asn Glu 35 40 45 Thr LeuArg Ile Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr 50 55 60 Glu GluIle Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Thr Val Gln 65 70 75 80 GlyGly Thr Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys 85 90 95 TyrIle Asp Gly Gln Lys Lys Lys Cys Gly Glu Glu Arg Arg Arg Val 100 105 110Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly Val Met Asn Thr 115 120125 Glu Trp Ile Ile Glu Ser 130 13 212 PRT Homo sapiens 13 Met Asn SerPhe Ser Thr Ser Ala Phe Gly Pro Val Ala Phe Ser Leu 1 5 10 15 Gly LeuLeu Leu Val Leu Pro Ala Ala Phe Pro Ala Pro Val Pro Pro 20 25 30 Gly GluAsp Ser Lys Asp Val Ala Ala Pro His Arg Gln Pro Leu Thr 35 40 45 Ser SerGlu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu Asp Gly Ile 50 55 60 Ser AlaLeu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met Cys Glu Ser 65 70 75 80 SerLys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys Met Ala 85 90 95 GluLys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr Cys Leu 100 105 110Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Tyr 115 120125 Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg Ala Val Gln 130135 140 Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln Lys Lys Ala Lys Asn145 150 155 160 Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn Ala SerLeu Leu 165 170 175 Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp MetThr Thr His 180 185 190 Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln SerSer Leu Arg Ala 195 200 205 Leu Arg Gln Met 210 14 177 PRT Homo sapiens14 Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile 1 510 15 Leu Val Leu Leu Pro Val Ala Ser Ser Asp Cys Asp Ile Glu Gly Lys 2025 30 Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile Asp Gln Leu 3540 45 Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe 5055 60 Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu Gly Met Phe 6570 75 80 Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys Met Asn Ser85 90 95 Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu Gly Thr Thr100 105 110 Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys Pro AlaAla 115 120 125 Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn LysSer Leu 130 135 140 Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu LysArg Leu Leu 145 150 155 160 Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile LeuMet Gly Thr Lys Glu 165 170 175 His 15 144 PRT Homo sapiens 15 Met LeuLeu Ala Met Val Leu Thr Ser Ala Leu Leu Leu Cys Ser Val 1 5 10 15 AlaGly Gln Gly Cys Pro Thr Leu Ala Gly Ile Leu Asp Ile Asn Phe 20 25 30 LeuIle Asn Lys Met Gln Glu Asp Pro Ala Ser Lys Cys His Cys Ser 35 40 45 AlaAsn Val Thr Ser Cys Leu Cys Leu Gly Ile Pro Ser Asp Asn Cys 50 55 60 ThrArg Pro Cys Phe Ser Glu Arg Leu Ser Gln Met Thr Asn Thr Thr 65 70 75 80Met Gln Thr Arg Tyr Pro Leu Ile Phe Ser Arg Val Lys Lys Ser Val 85 90 95Glu Val Leu Lys Asn Asn Lys Cys Pro Tyr Phe Ser Cys Glu Gln Pro 100 105110 Cys Asn Gln Thr Thr Ala Gly Asn Ala Leu Thr Phe Leu Lys Ser Leu 115120 125 Leu Glu Ile Phe Gln Lys Glu Lys Met Arg Gly Met Arg Gly Lys Ile130 135 140 16 178 PRT Homo sapiens 16 Met His Ser Ser Ala Leu Leu CysCys Leu Val Leu Leu Thr Gly Val 1 5 10 15 Arg Ala Ser Pro Gly Gln GlyThr Gln Ser Glu Asn Ser Cys Thr His 20 25 30 Phe Pro Gly Asn Leu Pro AsnMet Leu Arg Asp Leu Arg Asp Ala Phe 35 40 45 Ser Arg Val Lys Thr Phe PheGln Met Lys Asp Gln Leu Asp Asn Leu 50 55 60 Leu Leu Lys Glu Ser Leu LeuGlu Asp Phe Lys Gly Tyr Leu Gly Cys 65 70 75 80 Gln Ala Leu Ser Glu MetIle Gln Phe Tyr Leu Glu Glu Val Met Pro 85 90 95 Gln Ala Glu Asn Gln AspPro Asp Ile Lys Ala His Val Asn Ser Leu 100 105 110 Gly Glu Asn Leu LysThr Leu Arg Leu Arg Leu Arg Arg Cys His Arg 115 120 125 Phe Leu Pro CysGlu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn 130 135 140 Ala Phe AsnLys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu 145 150 155 160 PheAsp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile 165 170 175Arg Asn 17 199 PRT Homo sapiens 17 Met Asn Cys Val Cys Arg Leu Val LeuVal Val Leu Ser Leu Trp Pro 1 5 10 15 Asp Thr Ala Val Ala Pro Gly ProPro Pro Gly Pro Pro Arg Val Ser 20 25 30 Pro Asp Pro Arg Ala Glu Leu AspSer Thr Val Leu Leu Thr Arg Ser 35 40 45 Leu Leu Ala Asp Thr Arg Gln LeuAla Ala Gln Leu Arg Asp Lys Phe 50 55 60 Pro Ala Asp Gly Asp His Asn LeuAsp Ser Leu Pro Thr Leu Ala Met 65 70 75 80 Ser Ala Gly Ala Leu Gly AlaLeu Gln Leu Pro Gly Val Leu Thr Arg 85 90 95 Leu Arg Ala Asp Leu Leu SerTyr Leu Arg His Val Gln Trp Leu Arg 100 105 110 Arg Ala Gly Gly Ser SerLeu Lys Thr Leu Glu Pro Glu Leu Gly Thr 115 120 125 Leu Gln Ala Arg LeuAsp Arg Leu Leu Arg Arg Leu Gln Leu Leu Met 130 135 140 Ser Arg Leu AlaLeu Pro Gln Pro Pro Pro Asp Pro Pro Ala Pro Pro 145 150 155 160 Leu AlaPro Pro Ser Ser Ala Trp Gly Gly Ile Arg Ala Ala His Ala 165 170 175 IleLeu Gly Gly Leu His Leu Thr Leu Asp Trp Ala Val Arg Gly Leu 180 185 190Leu Leu Leu Lys Thr Arg Leu 195 18 219 PRT Homo sapiens 18 Met Cys ProAla Arg Ser Leu Leu Leu Val Ala Thr Leu Val Leu Leu 1 5 10 15 Asp HisLeu Ser Leu Ala Arg Asn Leu Pro Val Ala Thr Pro Asp Pro 20 25 30 Gly MetPhe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val 35 40 45 Ser AsnMet Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys 50 55 60 Thr SerGlu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser 65 70 75 80 ThrVal Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys 85 90 95 LeuAsn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala 100 105 110Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr 115 120125 Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys 130135 140 Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu145 150 155 160 Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn SerGlu Thr 165 170 175 Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe TyrLys Thr Lys 180 185 190 Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg IleArg Ala Val Thr 195 200 205 Ile Asp Arg Val Thr Ser Tyr Leu Asn Ala Ser210 215 19 132 PRT Homo sapiens 19 Met Ala Leu Leu Leu Thr Thr Val IleAla Leu Thr Cys Leu Gly Gly 1 5 10 15 Phe Ala Ser Pro Gly Pro Val ProPro Ser Thr Ala Leu Arg Glu Leu 20 25 30 Ile Glu Glu Leu Val Asn Ile ThrGln Asn Gln Lys Ala Pro Leu Cys 35 40 45 Asn Gly Ser Met Val Trp Ser IleAsn Leu Thr Ala Gly Met Tyr Cys 50 55 60 Ala Ala Leu Glu Ser Leu Ile AsnVal Ser Gly Cys Ser Ala Ile Glu 65 70 75 80 Lys Thr Gln Arg Met Leu SerGly Phe Cys Pro His Lys Val Ser Ala 85 90 95 Gly Gln Phe Ser Ser Leu HisVal Arg Asp Thr Lys Ile Glu Val Ala 100 105 110 Gln Phe Val Lys Asp LeuLeu Leu His Leu Lys Lys Leu Phe Arg Glu 115 120 125 Gly Arg Phe Asn 13020 114 PRT Homo sapiens 20 Asn Trp Val Asn Val Ile Ser Asp Leu Lys LysIle Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu TyrThr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys CysPhe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala SerIle His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser LeuSer Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys GluGlu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val HisIle Val Gln Met Phe Ile Asn 100 105 110 Thr Ser 21 252 PRT Homo sapiens21 Met Gly Val Leu Leu Thr Gln Arg Thr Leu Leu Ser Leu Val Leu Ala 1 510 15 Leu Leu Phe Pro Ser Met Ala Ser Met Ala Ala Ile Gly Ser Cys Ser 2025 30 Lys Glu Tyr Arg Val Leu Leu Gly Gln Leu Gln Lys Gln Thr Asp Leu 3540 45 Met Gln Asp Thr Ser Arg Leu Leu Asp Pro Tyr Ile Arg Ile Gln Gly 5055 60 Leu Asp Val Pro Lys Leu Arg Glu His Cys Arg Glu Arg Pro Gly Ala 6570 75 80 Phe Pro Ser Glu Glu Thr Leu Arg Gly Leu Gly Arg Arg Gly Phe Leu85 90 95 Gln Thr Leu Asn Ala Thr Leu Gly Cys Val Leu His Arg Leu Ala Asp100 105 110 Leu Glu Gln Arg Leu Pro Lys Ala Gln Asp Leu Glu Arg Ser GlyLeu 115 120 125 Asn Ile Glu Asp Leu Glu Lys Leu Gln Met Ala Arg Pro AsnIle Leu 130 135 140 Gly Leu Arg Asn Asn Ile Tyr Cys Met Ala Gln Leu LeuAsp Asn Ser 145 150 155 160 Asp Thr Ala Glu Pro Thr Lys Ala Gly Arg GlyAla Ser Gln Pro Pro 165 170 175 Thr Pro Thr Pro Ala Ser Asp Ala Phe GlnArg Lys Leu Glu Gly Cys 180 185 190 Arg Phe Leu His Gly Tyr His Arg PheMet His Ser Val Gly Arg Val 195 200 205 Phe Ser Lys Trp Gly Glu Ser ProAsn Arg Ser Arg Arg His Ser Pro 210 215 220 His Gln Ala Leu Arg Lys GlyVal Arg Arg Thr Arg Pro Ser Arg Lys 225 230 235 240 Gly Lys Arg Leu MetThr Arg Gly Gln Leu Pro Arg 245 250 22 200 PRT Homo sapiens 22 Met AlaPhe Thr Glu His Ser Pro Leu Thr Pro His Arg Arg Asp Leu 1 5 10 15 CysSer Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr 20 25 30 AlaLeu Thr Glu Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile 35 40 45 AsnLeu Asp Ser Ala Asp Gly Met Pro Val Ala Ser Thr Asp Gln Trp 50 55 60 SerGlu Leu Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr 65 70 75 80Arg Thr Phe His Val Leu Leu Ala Arg Leu Leu Glu Asp Gln Gln Val 85 90 95His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Ile His Thr Leu 100 105110 Leu Leu Gln Val Ala Ala Phe Ala Tyr Gln Ile Glu Glu Leu Met Ile 115120 125 Leu Leu Glu Tyr Lys Ile Pro Arg Asn Glu Ala Asp Gly Met Pro Ile130 135 140 Asn Val Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly LeuLys 145 150 155 160 Val Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser IleHis Asp Leu 165 170 175 Arg Phe Ile Ser Ser His Gln Thr Gly Ile Pro AlaArg Gly Ser His 180 185 190 Tyr Ile Ala Asn Asn Lys Lys Met 195 200 23181 PRT Homo sapiens 23 Ser Pro Leu Pro Ile Thr Pro Val Asn Ala Thr CysAla Ile Arg His 1 5 10 15 Pro Cys His Asn Asn Leu Met Asn Gln Ile ArgSer Gln Leu Ala Gln 20 25 30 Leu Asn Gly Ser Ala Asn Ala Leu Phe Ile LeuTyr Tyr Thr Ala Gln 35 40 45 Gly Glu Pro Phe Pro Asn Asn Leu Asp Lys LeuCys Gly Pro Asn Val 50 55 60 Thr Asp Phe Pro Pro Phe His Ala Asn Gly ThrGlu Lys Ala Lys Leu 65 70 75 80 Val Glu Leu Tyr Arg Ile Val Val Tyr LeuGly Thr Ser Leu Gly Asn 85 90 95 Ile Thr Arg Asp Gln Lys Ile Leu Asn ProSer Ala Leu Ser Leu His 100 105 110 Ser Lys Leu Asn Ala Thr Ala Asp IleLeu Arg Gly Leu Leu Ser Asn 115 120 125 Val Leu Cys Arg Leu Cys Ser LysTyr His Val Gly His Val Asp Val 130 135 140 Thr Tyr Gly Pro Pro Asp ThrSer Gly Lys Asp Val Phe Gln Lys Lys 145 150 155 160 Lys Leu Gly Cys GlnLeu Leu Gly Lys Tyr Lys Gln Ile Ile Ala Val 165 170 175 Leu Ala Gln AlaPhe 180 24 29 DNA Artificial Sequence Description of ArtificialSequencePCR Primer 24 catatgttcc caaccattcc cttatccag 29 25 33 DNAArtificial Sequence Description of Artificial SequencePCR Primer 25gggggatcct cactagaagc cacagctgcc ctc 33 26 39 DNA Artificial SequenceDescription of Artificial SequencePCR Primer 26 ccccggatcc gccaccatggatctctggca gctgctgtt 39 27 40 DNA Artificial Sequence Description ofArtificial SequencePCR Primer 27 ccccgtcgac tctagagcta ttaaatacgtagctcttggg 40 28 32 DNA Artificial Sequence Description of ArtificialSequencePCR Primer 28 cgcggatccg attagaatcc acagctcccc tc 32 29 66 DNAArtificial Sequence Description of Artificial SequencePCR Primer 29ccccctctag acatatgaag aagaacatcg cattcctgct ggcatctatg ttcgttttct 60ctatcg 66 30 65 DNA Artificial Sequence Description of ArtificialSequencePCR Primer 30 gcatctatgt tcgttttctc tatcgctacc aacgcttacgcattcccaac cattccctta 60 tccag 65 31 62 DNA Artificial SequenceDescription of Artificial SequencePCR Primer 31 gcagtggcac tggctggtttcgctaccgta gcgcaggcct tcccaaccat tcccttatcc 60 ag 62 32 59 DNAArtificial Sequence Description of Artificial SequencePCR Primer 32ccccgtcgac acatatgaag aagacagcta tcgcgattgc agtggcactg gctggtttc 59 3336 DNA Artificial Sequence Description of Artificial SequencePCR Primer33 ctgcttgaag atctgcccac accgggggct gccatc 36 34 24 DNA ArtificialSequence Description of Artificial SequencePCR Primer 34 gtagcgcaggccttcccaac catt 24 35 39 DNA Artificial Sequence Description ofArtificial SequencePCR Primer 35 ctgcttgaag atctgcccag tccgggggcagccatcttc 39 36 51 DNA Artificial Sequence Description of ArtificialSequencePCR Primer 36 gggcagatct tcaagcagac ctacagcaag ttcgactgcaactcacacaa c 51 37 34 DNA Artificial Sequence Description of ArtificialSequencePCR Primer 37 cgcggtaccc gggatccgat tagaatccac agct 34 38 36 DNAArtificial Sequence Description of Artificial SequencePCR Primer 38gggcagatct tcaagcagac ctactgcaag ttcgac 36 39 42 DNA Artificial SequenceDescription of Artificial SequencePCR Primer 39 cgcggtaccg gatccttagcagaagccaca gctgccctcc ac 42 40 24 DNA Artificial Sequence Description ofArtificial SequencePCR Primer 40 gtagcgcagg ccttcccaac catt 24 41 40 DNAArtificial Sequence Description of Artificial SequencePCR Primer 41ccccgtcgac tctagagcca ttagatacaa agctcttggg 40

What is claimed is:
 1. A cysteine variant of granulocytecolony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue issubstituted for at least one amino acid located in at least one regionof granulocyte colony-stimulating factor selected from the groupconsisting of: the A-B loop, the B-C loop, the first three or last threeamino acids in helix B, the first three or last three amino acids inhelix C, the first three or last three amino acids in helix D, and theamino acids following helix D; wherein said variant has biologicalactivity in vitro as measured by proliferation of a cell line thatproliferates in response to granulocyte colony-stimulating factor. 2.The cysteine variant according to claim 1, wherein a cysteine residue issubstituted for at least one amino acid selected from the groupconsisting of: K40, S53, G55, W58, A59, P60, S62, S63, P65, S66, Q67,A68, Q70, A72, Q90, A91, E93, G94, S96, E98, G100, A143, Q145, Q173 andP174.
 3. A cysteine variant of granulocyte colony-stimulating factor ofSEQ ID NO:6, wherein said variant contains T133 and wherein a cysteineresidue is substituted for at least one amino acid located in at leastone region of granulocyte colony-stimulating factor selected from thegroup consisting of: the A-B loop, the B-C loop, the C-D loop, the firstthree or last three amino acids in helix B, the first three or lastthree amino acids in helix C, the first three or last three amino acidsin helix D, and the amino acids following helix D; wherein said varianthas biological activity in vitro as measured by proliferation of a cellline that proliferates in response to granulocyte colony-stimulatingfactor.
 4. The cysteine variant according to claim 3, wherein a cysteineresidue is substituted for at least one amino acid selected from thegroup consisting of: K40, S53, G55, W58, A59, P60, S62, S63, P65, S66,Q67, A68, Q70, A72, Q90, A91, E93, G94, S96, E98, G100, G125, M126,A127, A129, Q131, Q134, G135, A136, A139, A141, S142, A143, Q145, Q173and P174.
 5. A cysteine variant of granulocyte colony-stimulating factorof SEQ ID NO:6, wherein a cysteine residue is substituted for at leastone amino acid located in the region of granulocyte colony-stimulatingfactor following helix D; wherein said variant has biological activityin vitro as measured by proliferation of a cell line that proliferatesin response to granulocyte colony-stimulating factor.
 6. The cysteinevariant according to claim 5, wherein a cysteine residue is substitutedfor Q173.
 7. The cysteine variant according to claim 5, wherein acysteine residue is substituted for P174.
 8. A cysteine variant ofgranulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteineresidue is substituted for at least one amino acid located in the A-Bloop of granulocyte colony-stimulating factor; wherein said variant hasbiological activity in vitro as measured by proliferation of a cell linethat proliferates in response to granulocyte colony-stimulating factor.9. The cysteine variant according to claim 8, wherein a cysteine residueis substituted for at least one amino acid selected from the groupconsisting of K40, S53, G55, W58, A59, P60, S62, S63, P65, S66, Q67,A68, and Q70.
 10. The cysteine variant according to claim 8, wherein acysteine residue is substituted for W58.
 11. The cysteine variantaccording to claim 8, wherein a cysteine residue is substituted for A68.12. A cysteine variant of granulocyte colony-stimulating factor of SEQID NO:6, wherein a cysteine residue is substituted for at least oneamino acid located in the B-C loop of granulocyte colony-stimulatingfactor; wherein said variant has biological activity in vitro asmeasured by proliferation of a cell line that proliferates in responseto granulocyte colony-stimulating factor.
 13. The cysteine variantaccording to claim 12, wherein a cysteine residue is substituted for atleast one amino acid selected from the group consisting of E93, G94,S96, and E98.
 14. The cysteine variant according to claim 12, wherein acysteine residue is substituted for E93.
 15. The cysteine variantaccording to claim 12, wherein a cysteine residue is substituted forE98.
 16. A cysteine variant of granulocyte colony-stimulating factor ofSEQ ID NO:6, wherein a cysteine residue is substituted for at least oneamino acid located in the C-D loop of granulocyte colony-stimulatingfactor; wherein said variant has biological activity in vitro asmeasured by proliferation of a cell line that proliferates in responseto granulocyte colony-stimulating factor.
 17. The cysteine variantaccording to claim 16, wherein a cysteine residue is substituted for atleast one amino acid selected from the group consisting of G125, M126,A127, A129, Q131, Q134, G135, A136, A139, A141, and S142.
 18. Thecysteine variant according to claim 16, wherein a cysteine residue issubstituted for A129.
 19. The cysteine variant according to claim 16,wherein a cysteine residue is substituted for Q131.
 20. The cysteinevariant according to claim 16, wherein a cysteine residue is substitutedfor T133.
 21. The cysteine variant according to claim 16, wherein acysteine residue is substituted for Q134.
 22. The cysteine variantaccording to claim 16, wherein a cysteine residue is substituted forA136.
 23. The cysteine variant according to claim 16, wherein a cysteineresidue is substituted for A139.
 24. The cysteine variant according toclaim 16, wherein a cysteine residue is substituted for A141.
 25. Acysteine variant of granulocyte colony-stimulating factor of SEQ IDNO:6, wherein said variant contains T133 and wherein a cysteine residueis substituted for at least one amino acid located in the C-D loop ofgranulocyte colony-stimulating factor; wherein said variant hasbiological activity in vitro as measured by proliferation of a cell linethat proliferates in response to granulocyte colony-stimulating factor.26. The cysteine variant according to claim 25, wherein a cysteineresidue is substituted for at least one amino acid selected from thegroup consisting of G125, M126, A127, A129, Q131, Q134, G135, A136,A139, A141, and S142.
 27. A cysteine variant of granulocytecolony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue issubstituted for at least one of the first three amino acids in helix Aof granulocyte colony-stimulating factor; wherein said variant hasbiological activity in vitro as measured by proliferation of a cell linethat proliferates in response to granulocyte colony-stimulating factor.28. The cysteine variant according to claim 27, wherein a cysteineresidue is substituted for at least one amino acid selected from thegroup consisting of Q11 and S12.
 29. A cysteine variant of granulocytecolony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue issubstituted for T38; wherein said variant has biological activity invitro as measured by proliferation of a cell line that proliferates inresponse to granulocyte colony-stimulating factor.
 30. A cysteinevariant of granulocyte colony-stimulating factor of SEQ ID NO:6, whereina cysteine residue is substituted for at least one amino acid located inat least one region of granulocyte colony-stimulating factor selectedfrom the group consisting of the first three amino acids in helix B andthe last three amino acids in helix B; wherein said variant hasbiological activity in vitro as measured by proliferation of a cell linethat proliferates in response to granulocyte colony-stimulating factor.31. The cysteine variant according to claim 30, wherein a cysteineresidue is substituted for at least one amino acid selected from thegroup consisting of A72, Q90 and A91.
 32. A cysteine variant ofgranulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteineresidue is substituted for at least one amino acid located in at leastone region of granulocyte colony-stimulating factor selected from thegroup consisting of the first three amino acids in helix C and the lastthree amino acids in helix C; wherein said variant has biologicalactivity in vitro as measured by proliferation of a cell line thatproliferates in response to granulocyte colony-stimulating factor. 33.The cysteine variant according to claim 33, wherein a cysteine residueis substituted for G100.
 34. A cysteine variant of granulocytecolony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue issubstituted for at least one amino acid located in at least one regionof granulocyte colony-stimulating factor selected from the groupconsisting of the first three amino acids in helix D and the last threeamino acids in helix D; wherein said variant has biological activity invitro as measured by proliferation of a cell line that proliferates inresponse to granulocyte colony-stimulating factor.
 35. The cysteinevariant according to claim 34, wherein a cysteine residue is substitutedfor at least one amino acid selected from the group consisting of A143and Q145.
 36. A cysteine variant of granulocyte colony-stimulatingfactor of SEQ ID NO:6, wherein a cysteine residue is substituted for atleast one amino acid of granulocyte colony-stimulating factor selectedfrom the group consisting of P2, S7, S8, L9 and P10; wherein saidvariant has biological activity in vitro as measured by proliferation ofa cell line that proliferates in response to granulocytecolony-stimulating factor.
 37. The cysteine variant according to claim36, wherein a cysteine residue is substituted for P2.
 38. The cysteinevariant according to claim 36, wherein a cysteine residue is substitutedfor S7.
 39. The cysteine variant according to any one of claims 1, 3, 5,8, 12, 16, 25, 27, 29, 30, 32, 34 or 36, wherein a non-cysteine aminoacid is substituted for C17.
 40. The cysteine variant according to claim39, wherein the non-cysteine amino acid substituted for C17 is selectedfrom the group consisting of serine and alanine.
 41. The cysteinevariant according to any one of claims 1, 3, 5, 8, 12, 16, 25, 27, 29,30, 32, 34 or 36, wherein the substituted cysteine residue is modifiedwith a cysteine-reactive moiety.
 42. The cysteine variant according toany one of claims 1, 3, 5, 8, 12, 16, 25, 27, 29, 30, 32, 34 or 36,wherein the substituted cysteine residue is modified with acysteine-reactive moiety, and wherein a non-cysteine amino acid issubstituted for C17.
 43. The cysteine variant according to claim 42,wherein the non-cysteine amino acid substituted for C17 is selected fromthe group consisting of serine and alanine.
 44. The cysteine variantaccording to any one of claims 1, 3, 5, 8, 12, 16, 25, 27, 29, 30, 32,34 or 36, wherein the substituted cysteine residue is modified withpolyethylene glycol.
 45. The cysteine variant according to any one ofclaims 1, 3, 5, 8, 12, 16, 25, 27, 29, 30, 32, 34 or 36, wherein thesubstituted cysteine residue is modified with polyethylene glycol, andwherein a non-cysteine amino acid is substituted for C17.
 46. Thecysteine variant according to claim 45, wherein the non-cysteine aminoacid substituted for C17 is selected from the group consisting of serineand alanine.
 47. The cysteine variant according to any one of claims 1,3, 5, 8, 12, 16, 25, 27, 29, 30, 32, 34 or 36, wherein said cysteinevariant is modified with at least one polyethylene glycol.
 48. Thecysteine variant according to any one of claims 1, 3, 5, 8, 12, 16, 25,27, 29, 30, 32, 34 or 36, wherein said cysteine variant is modified withat least one polyethylene glycol, and wherein a non-cysteine amino acidis substituted for C17.
 49. The cysteine variant according to claim 48,wherein the non-cysteine amino acid substituted for C17 is selected fromthe group consisting of serine and alanine.
 50. A cysteine variant ofgranulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteineresidue is substituted for at least one amino acid selected from thegroup consisting of T1, L3, G4, P5, and A6, and wherein the substitutedcysteine residue is modified with a cysteine-reactive moiety; whereinsaid variant has biological activity in vitro as measured byproliferation of a cell line that proliferates in response togranulocyte colony-stimulating factor.
 51. The cysteine variantaccording to claim 50, wherein a cysteine residue is substituted for T1.52. The cysteine variant according to claim 50, wherein a cysteineresidue is substituted for L3.
 53. The cysteine variant according toclaim 50, wherein a cysteine residue is substituted for A6.
 54. Acysteine variant of granulocyte colony-stimulating factor of SEQ IDNO:6, wherein a cysteine residue is substituted for at least one aminoacid selected from the group consisting of T1, L3, G4, P5, and A6, andwherein the substituted cysteine residue is modified with a polyethyleneglycol; wherein said variant has biological activity in vitro asmeasured by proliferation of a cell line that proliferates in responseto granulocyte colony-stimulating factor.
 55. The cysteine variantaccording to claim 54, wherein a cysteine residue is substituted for T1.56. The cysteine variant according to claim 54, wherein a cysteineresidue is substituted for L3.
 57. The cysteine variant according toclaim 54, wherein a cysteine residue is substituted for A6.
 58. Thecysteine variant according to any one of claims 50 or 53, wherein anon-cysteine amino acid is substituted for C17.
 59. The cysteine variantaccording to claim 58, wherein the non-cysteine amino acid substitutedfor C17 is selected from the group consisting of serine and alanine.